JP4300029B2 - Method and apparatus for gel-free qualitative and quantitative proteomic analysis and use thereof - Google Patents

Abstract

Methods and apparatus for qualitative and quantitative proteome analysis are provided. The methods and apparatus allow for the isolation of a subset of peptides out of complex mixtures of peptides. The isolation is based on a specific chemical and/or enzymatic alteration of one or more types of peptides. This alteration modifies the biophysical, chemical or any other biochemical property of the affected types of peptides (e.g., net electrical charge and/or hydrophobicity) in such way that the altered peptides can be separated from the unaltered peptides.

Description

SUMMARY OF THE INVENTION Methods and apparatus for qualitative and quantitative proteomic analysis of the invention are provided. The method and apparatus make it possible to isolate a peptide subset from a complex mixture of peptides. The isolation is based on specific chemical and / or enzymatic modification of one or more types of peptides. This modification is performed in such a way that the modified peptide can be separated from the unmodified peptide in such a way that the biophysical, chemical or any other biochemical properties of the affected type of peptide (eg net charge and / or Hydrophobicity) is modified.

  In one embodiment, this modification is applied between the first chromatographic separation of the complex peptide mixture and the modified complex mixture using the same type of chromatographic separation in the first and second separations. The “Same type of chromatographic separation” means that both the first and second chromatographic separations are based on hydrophobicity or both are based on ion exchange. Therefore, in the method of the present invention, a first separation step is used in which the complex peptide mixture is separated into fractions based on the elution or movement pattern. Each fraction is then subjected to a specific modification reaction that can be driven chemically or enzymatically or chemically and enzymatically. At this time, each fraction is again subjected to the second separation. Based on i) the type of modification and ii) the separation conditions, the modified subset of peptides within each fraction will elute or migrate in a separated state from the unmodified peptides. Furthermore, the present invention selectively and using either a single column system or a multiple column system of the same or similar columns, which can be implemented in either parallel only, serial only or serial / parallel combination mode. An apparatus for efficiently carrying out the method is provided. The isolated peptide can then be released gradually and sequentially and transferred to an analytical system for identification.

BACKGROUND OF THE INVENTION The proteome has been defined as the total complement of proteins expressed by cells, tissue types or organisms, and proteomics are expressed at a given point in time or under certain environmental conditions. This is a supplemental study done. Such comprehensive analysis requires that thousands of proteins are routinely identified and characterized from a single sample. Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) is considered to be an important tool for proteomics that produces separations that display up to thousands of protein spots on a 2D gel. Proteins in the gel can be detected to some extent by using various stains that allow quantification and comparison between gels from different samples. Protein identification can be achieved, for example, by excising the protein spot and digesting the spot with a protease of known specificity. Peptides resulting from such cleavage have a specific mass that can then be determined by mass spectrometry. These data are compared to the mass of the peptide in the database. The latter mass is, for example, in silico data obtained by calculating the molecular weight of each protein and its fragment fragments starting from DNA sequence data. When the mass of a peptide that is accurately determined spectroscopically matches the mass of the peptide in a computer, this is often sufficient to annotate the peptide to its parent protein. Alternatively, conversely, a particular protein in a sample can be identified by identifying one or more of its constituent peptide fragments (so-called peptide mass fingerprinting).

  However, 2D-PAGE is sequential and labor intensive and difficult to automate. In addition, certain classes of proteins, such as membrane proteins, very large and small proteins, and very acidic or basic proteins, are difficult to analyze using this method. Another important drawback is that proteins that are highly abundant in analysis are rare because regulatory proteins (eg, transcription factors and protein kinases) that are relatively abundant in the analysis of whole cell lysates are rarely detected. It is in the point of deviating toward.

  Because of these shortcomings, scientists have sought alternative approaches for analyzing proteomes that do not require each protein to be purified to homogeneity. These techniques are referred to herein as “gel-free systems” and do not use a gel separation process. The peptide mass fingerprint approach taught us that proteins can be identified based on the mass of one or more of its constituent peptides. One approach to analyze proteins in a biological sample was to proteolyze the proteins and determine the resulting peptide mass. As long as the sample contains only a small amount of different proteins, the resulting number of peptides is small and can be identified by chromatographic separation of the peptides and subsequent analysis by mass spectrometry. In most complex biological samples, proteolysis of the protein will produce thousands of peptides, which exceeds the degradation capability of any known chromatographic system. The result is co-elution of individual peptides and thus poor separation and isolation. Furthermore, the resolution of mass spectrometry coupled with such chromatography is not sufficient to adequately determine the mass of individual peptides. One approach to improve the resolution of complex mixtures of peptides is to use multidimensional chromatography, such as the recently described process of direct analysis of large protein complexes (DALPC) (Link et al. (1999)). The DALPC process uses independent physical properties and hydrophobicity of charge to resolve complex peptide mixtures by a combination of strong cation exchange and reverse phase chromatography. Although this method improves the separation of complex mixtures into their individual components, the resolution of this approach is still far from sufficient to reproducibly identify component peptides in biological samples. . A further disadvantage of the DALPC method is that it is incompatible with the analysis of low abundance proteins and cannot be used quantitatively.

  A recently described second approach, ICAT-method, is based on the use of a combination of a new chemical reagent called isotope-coded affinity tag (ICATS) and tandem mass spectrometry (Gygi et al (1999)). . The ICAT-method is based on the modification of cysteine-containing proteins with iodoacetate derivatives carrying a biotin label. After enzymatic cleavage of the modified protein into peptides, only the cysteine-modified labeled peptides are pulled down with streptavidin-coated beads in an affinity purification step. The affinity purification process reduces the complex of the original peptide mixture and makes separation of component peptides by liquid chromatography combined with mass spectrometry a more feasible realistic goal. However, the disadvantage is that the affinity purification process generally requires a larger amount of starting material due to material loss during the purification process. Furthermore, ICAT label is a relatively large modification (˜500 Da) that stays on each peptide throughout the MS analysis and complicates the database search algorithm, especially for small peptides. The method is similarly useless for proteins that do not contain any cysteine residues. Moreover, because of the affinity purification process, the modified peptide is produced at once and released in the form of a so-called compressed mixture. This means that there is no optimal chromatographic separation at all and the efficiency of detection of modified peptides by mass spectrometry is relatively low. Similarly, in two other publications (Geng et al., 2000 and Ji et al., 2000), affinity chromatography is used to select peptide subsets and is simply used to identify the corresponding parent protein. A separated signature peptide is used.

  The present invention describes a novel gel-free method for qualitative and quantitative proteomic analysis that does not require multidimensional chromatography and does not use affinity tags. This method is very adaptable and can be applied to a large number of different peptide classes, even to biological samples containing only a few cells.

Detailed Description of the Invention The present invention provides methods and apparatus for the isolation and identification of a subset of peptides from a complex peptide mixture.

  The method comprises the steps of modifying a selected population of peptides in such a way that the chromatographic action of the modified peptide in the second chromatographic separation differs from the chromatographic action of its unmodified version. Utilizing a combination of two chromatographic separations of the same type separated.

  In order to isolate a subset of peptides from a protein peptide mixture, the present invention is applicable in two modes of action. In the first mode, a small number of peptides in a protein peptide mixture are modified and a subset of modified peptides is isolated. In this mode of action, the modified peptide is called a flagged peptide. In the second mode, multiple peptides in a protein peptide mixture are modified and a subset of unmodified peptides is isolated. In this mode of action, the unmodified peptide is called the identified peptide.

  In one embodiment, the present invention provides a method for isolating a subset of peptides from a protein peptide mixture, the step of (a) separating the protein peptide mixture into peptide fractions by chromatography; (b) within each fraction Modifying at least one amino acid of at least one of the peptides chemically or enzymatically or chemically and enzymatically to produce a subset of modified peptides, and (c) chromatographically Isolating the modified or so-called flagged peptide from the fraction, providing a method wherein the chromatography of steps (a) and (c) is carried out with the same type of chromatography.

In another embodiment, the present invention provides a method for isolating a subset of peptides from a protein peptide mixture, comprising:
(A) first separating the protein peptide mixture into peptide fractions by chromatography; (b) chemically or enzymatically or chemically at least one amino acid of the majority of the peptides in each fraction; And enzymatically modifying to produce a subset of unmodified peptides, and (c) isolating said unmodified or so-called identified peptides by a second chromatography, wherein the first and second It provides a method in which the chromatography of the separation step is carried out with the same type of chromatography.

  The same type of chromatography means that the chromatography type is the same in the first and second separations. The type of chromatography is for example based on the hydrophobicity of the peptide in both separations. Similarly, the type of chromatography can be based on the use of peptide charge and ion exchange chromatography in both steps. In yet another variation, the chromatographic separation is based on size exclusion chromatography or any other type of chromatography in both steps.

  The first chromatographic separation prior to modification is referred to below as the “primary run” or “primary chromatography step” or “primary chromatographic separation” or “run 1”. The second chromatographic separation of the modified fraction is hereinafter referred to as “secondary run” or “secondary chromatography step” or “secondary chromatographic separation” or “run 2”.

  In preferred embodiments of the invention, the chromatographic conditions for the primary and secondary runs are the same; or are substantially similar to those skilled in the art. Substantially similar is, for example, that the chromatographic conditions lead to the elution of modified peptides that are completely different from the unmodified peptides in a predictable manner for all fractions recovered from the primary run. Means that slight changes in flow rate and / or gradient and / or temperature and / or pressure and / or chromatographic beads and / or solvent composition are allowed between primary and secondary runs. To do.

  As used herein, a “protein peptide mixture” is typically a complex peptide mixture obtained as a result of cleaving a sample containing protein. Such samples are typically, without limitation, from any complex mixture or single cell of protein, such as prokaryotic or eukaryotic cell lysates, or from specific organelle fractions, biopsies, laser-captured incised cells. Any complex mixture of isolated proteins or any large protein complex such as ribosome, virus, etc. If such protein samples are divided into peptides, it can be expected that they can contain up to 1000, 5000, 10,000, 20,000, 30,000, 100,000 or more different peptides. However, in certain cases, the “protein peptide mixture” may also be derived directly from body fluids or more generally from any solution of biological origin. For example, urine contains, in addition to protein, a highly complex peptide mixture resulting from proteolytic degradation of in vivo proteins from which peptides are removed by the kidneys. Yet another example of a protein peptide mixture is a mixture of peptides present in cerebrospinal fluid.

More generally speaking, the present invention applies to any complex peptide mixture. As used herein, “complex peptide mixture” means a mixture of 100 or more different peptides, typically 500 or more, and more typically 1000 or more. In the present invention, the terms “protein peptide mixture” and “complex peptide mixture” are used interchangeably.
Similarly, as used herein, “subset of peptides” from a protein peptide mixture means a fraction of the total number of peptides present in the protein peptide mixture. Such fractions will certainly be less than 50% of the initial peptide number, typically less than 20%, and more typically less than 10% of the initial peptide number in the protein peptide mixture.

  The terms “alter” or “altered” or “altered” as used herein with respect to peptides refer to peptides with the express intention of altering the chromatographic behavior of peptides containing modified amino acids. It means introducing specific modifications into amino acids. As used herein, the term “modified peptide” is a peptide that contains an amino acid that has been modified as a result of the modification.

  Such alteration can be a stable chemical or enzymatic modification. Such modifications can also introduce temporary interactions with amino acids. Typically, one modification is a covalent reaction, but the modification may consist of complex formation, provided that the complex is sufficiently stable during the chromatography process.

  Typically, a modification, unlike its unmodified version, results in a change in hydrophobicity such that the modified peptide migrates in hydrophobic chromatography. Alternatively, the modification, unlike its unmodified version, results in a change in the net charge of the peptide in such a way that the modified peptide migrates within ion exchange chromatography, such as anion exchange or cation exchange chromatography. Bring. Similarly, modification results in any other biochemical, chemical or biophysical change in the peptide such that the modified peptide moves within the chromatographic separation, unlike its unmodified version. There is a possibility to bring. The term “migrate differently” means that a particular modified peptide elutes with a different elution time compared to the elution time of the same unmodified peptide.

  Modifications can be obtained by chemical and enzymatic reactions or a combination thereof. A non-limiting list of chemical reactions includes alkylation, acetylation, nitrosylation, oxidation, hydroxylation, methylation, reduction, and the like. A non-limiting list of enzymatic reactions includes treatment of peptides with phosphatases, acetylases, glycosidases, or other enzymes that modify co-translational or post-translational modifications present on the peptide. A chemical modification can involve a single chemical reaction, but can also involve multiple reactions (eg, β-elimination reactions and oxidation), such as two sequential reactions to increase modification efficiency. It can also be included. Similarly, an enzymatic modification can include one or more enzymatic reactions.

  Another essential feature of the modification within the present invention is that the modification allows the isolation of a peptide subset from a protein peptide mixture. Chemical and / or enzymatic reactions that result in global modification of all peptides within a protein-peptide mixture do not allow isolation of peptide subsets. Thus, the modification must modify a particular peptide population within the protein peptide mixture to allow isolation of a subset of peptides when it is applied between two chromatographic separations of the same type.

  One approach to be able to isolate a peptide subset consisting of flagged peptides is to target the modification to a rare amino acid. Dilute amino acids are considered as amino acids that are not overly abundant in the complex peptide mixture. For example, if a specific amino acid occupies an excessively abundant portion of a peptide mixture (eg, 50% or more of the peptide), too many peptides will be selected and the flagged peptide Efficient separation and isolation will again be impossible. Preferably, a proportion of less than 30%, 25%, 20%, 15%, 10%, 5%, 1%, 0.1%, 0.01% or more of the peptide from the complex peptide is selected .

  In one preferred embodiment, the specific amino acid selected for modification comprises one of the following amino acids: methionine (Met), cysteine (Cys), histidine (His), tyrosine (Tyr) ), Lysine (Lys), tryptophan (Trp), arginine (Arg), proline (Pro) or phenylalanine (Phe).

Alternatively, the alteration is specifically targeted to a population of amino acids that carry simultaneous or post-translational modifications. Examples of such simultaneous or post-translational modifications include glycosylation, phosphorylation, acetylation, formylation, ubiquitination, pyroglutamylation, hydroxylation, nitrosylation, ε-N-acetylation, sulfation, NH ₂ -There is end-blocking. Examples of modified amino acids modified to isolate peptide subsets according to the present invention include phosphoserine (phospho-Ser), phospho-threonine (phospho-Thr), phospho-aspartate (phospho-Asp), or There is acetyl-lysine.

A further non-limiting list of amino acids that can be modified and used to select peptide subsets includes other modified amino acids (eg, glycosylated amino acids), artificially incorporated D-amino acids, seleno Examples include amino acids and amino acids carrying unnatural isotopes. Modifications can also target one or more amino acids added to certain amino acids in vitro or specific residues on the modification (eg, free NH ₂ -terminal groups).

  In a preferred embodiment of the invention, the selected amino acid to be modified must be present in the majority of proteins in the sample that are rare but nevertheless contain the protein. This embodiment allows for the isolation of flagged peptides that account for the majority of the protein in the sample. Most proteins should contain at least one, preferably two, three or a limited number of residues of selected amino acids. For example, cysteine is a rare amino acid and only 14.55% of open reading frames from E. coli genomic sequences and / or proteins do not contain Cys. These numbers are 11.34% for Trp, 4.12% for His, and 0.32% for Met, respectively. The latter number increases to 3.17% after deletion of the initiator methionine, which is frequently processed. A similar theoretical analysis using other genomic sequences stored in the Swiss-Prot database (Release 39.0) is summarized in Table I. These studies reveal that, among the rare amino acids, methionine exhibits excellent protein representativeness among various species such as mammals, yeast and E. coli. More than 96% of the protein contains at least one internal methionine. This number is consistently lower for cysteines present in 85% to 95% of the predicted proteins, depending on the organism. These values show that fragment corrected SWISS-PROT with the sequence of 72,101 indicating that 2.3% of the protein did not contain Met, while 12.8% of the protein lacked Cys. And the results of early studies based on the SWISS-NEW annotated database (Vuong et al., 2000). Furthermore, these studies revealed that Met is more uniformly distributed than Cys on different proteins.

  Methionine is an amino acid that is preferably targeted for modification in the present invention. Cysteine, histidine and tryptophan are likewise preferred amino acids. Other less frequently found amino acids such as lysine, phenylarinin and tyrosine can also be used in the present invention. The selection of the amino acid to be modified also depends on the complex and origin of the sample protein (eg plant, animal, bacteria or virus). For example, in plants, methionine is a less representative amino acid, so it is more appropriate to select an amino acid such as cysteine as the specific amino acid to be modified.

  Alternatively, a specific chemical and / or enzymatic reaction has specificity for one or more amino acid residues (eg, both phosphoserine and phosphothreonine or a combination of methionine and cysteine) from a protein peptide mixture. Allows separation of peptide subsets. However, typically, the number of selected amino acids to be modified will be 1, 2 or 3. In another aspect, two different types of selected amino acids can be modified in a protein peptide mixture, and a flagged subset of peptides containing one or both modified amino acids is simply Can be separated. In yet another aspect, the same peptide mixture is first modified on one amino acid, a flagged subset of peptides is isolated, and a second modification is performed on the remaining previously modified sample; Another subset of flagged peptides can be isolated.

The present invention requires that the modification be effective for each of the peptide fractions from the primary run. Thereby, within each fraction obtained from the primary chromatography step, the flagged peptide must migrate quite differently from the unmodified peptide in the secondary chromatography step. Modification of one amino acid in the flagged peptide induces a shift in the elution of the flagged peptide. Depending on the type of modification applied, the shift can be caused by changes in the affinity and / or net charge, hydrophobicity of the flagged peptide ligand (eg, metal ion). This shift is called δp and is specific for all individual flagged peptides. In examples of hydrophobic changes, the δp value can be expressed as a change in hydrophobic moment or as a percentage of organic solvent in a chromatographic run, but most practically, for a given chromatographic / electrical It can be expressed in units of time under electrophoresis conditions. Thereby, δp is not necessarily the same for all flagged peptides and exists between δmax and δmin (see FIG. 1). δp is affected by a certain number of factors such as the nature of the induced modification, the nature of the column stationary phase, the mobile phase (buffer, solvent), temperature, etc. Considering all δp values together, the extreme values of δmax and δmin are limited (see FIG. 1). Let t ₁ and t _{2 be} the time limiting the beginning and end of the flagged peptide interval, and t ₃ -t _{4 be} the time surrounding the fraction taken from the primary run, then δmin at t ₃ -t ₂ (Minimum shift) is determined, while δmax (maximum shift) is determined by t ₄ −t ₁ . Window w ₁ is the fraction taken from the primary run (w ₁ = t ₄ −t ₃ ). Window w ₂ is a window in which FG will be eluted (w ₂ = t ₂ −t ₁ ). Thereby, δmin = t ₃ −t ₂ ; δmax = t ₄ −t ₁ ; w ₁ = δmax + t ₁ −δmin−t ₂ and w ₂ = t ₂ −t ₁ = δmax−δmin−w ₁ . The key elements in the sorting process are the elution of δmin, which limits the distance between the least shifted and unmodified flagged peptides within a given fraction and the flagged components. W ₂ which is the time window for The term “sorted” is equivalent to the term “isolated” in the present invention.

δmin must be sufficient to avoid the flagged peptide eluting within window w ₁ (and thus overlapping with the unmodified peptide); Should be applied to all fractions recovered from Preferentially, δmin should be w ₁ or higher to minimize the overlap between the flagged and unmodified peptides. For example, if w ₁ = 1 minute, δmin should be greater than 1 minute if possible.

Avoiding overlapping or co-elution of flagged peptides improves the possibility of identifying the optimal number of individual peptides. From this point of view, the size of window w ₂ affects the number of identifiable peptides. When the value of w ₂ is increased, restoring elution time of peptide flagged is obtained as a result, better isolation of peptides flagged, and to analyzers such as mass spectrometer anchoring compound Provides a better opportunity for analysis by presenting progressively. Although window w ₂ can be smaller than w ₁ , in a preferred embodiment, w ₂ is larger than w ₁ . For example, if w ₁ = 1 minute, w ₂ can be 1 minute or more. The size of w ₂ and the values of δmin and δmax are preferably the same or very similar for all fractions collected from the primary run. However, it is self-evident that slight contamination of the unmodified peptide within the elution window of the flagged peptide is undesirable but acceptable.

Δmin to obtain optimal separation of peptides flagged from unmodified peptide in each of the primary run fraction, the operation of the values of δmax and w ₂ are part of the present invention, among others, modifications Including the correct combination of amino acid (s) selected for, type of modification and chromatographic conditions (column type, buffer, solvent, etc.).

While the hydrophilic shift aspect has been implemented above, a similar description could be provided as well, in which a hydrophobic shift is induced to separate the flagged peptide from the unmodified peptide. Here t ₃ and t ₄ define the window w _{1 from} which the unmodified peptide elutes, while t ₅ and t ₆ define the window w _{2 from} which the flagged peptide elutes. Maximum hydrophobic shift δmax = t ₆ −t ₃ , minimum shift = t ₅ −t ₄ (FIG. 1 c). It will be appreciated that a similar calculation for the conditions for pooling fractions can be used.

  It will be apparent to those skilled in the art that the same approach can be applied to isolate peptides that have been flagged, for example, by ion exchange chromatography or other types of chromatography.

  In one embodiment, the present invention provides a method for isolating a methionine-containing peptide from a protein peptide mixture, wherein (a) separating the protein peptide mixture into fractions by a primary run, (b) each peptide fraction. And (c) isolating a methionine-containing peptide flagged by a secondary run. In certain embodiments, the primary run and secondary run are chromatographic separations based on hydrophobicity, and modification of methionine induces a hydrophilic shift of the flagged methionine-containing peptide. In a further specific embodiment, hydrophobic chromatography is performed on a reverse phase column and the modification of methionine is obtained with mild oxidation. In yet another embodiment, the primary and secondary runs are based on ion exchange chromatography and the modification of methionine is a chemical reaction with an alkyl halide such as methyl iodide. This reaction induces a charge change on the flagged peptide, allowing the flagged peptide to be separated in a secondary run on the ion exchange column.

  In another embodiment, the present invention provides a method for isolating phosphorylated peptides from a protein peptide mixture, wherein (a) separating the protein peptide mixture into fractions by a primary run; (b) A method is provided comprising the steps of enzymatically and / or chemically modifying the phosphopeptide in each fraction and (c) isolating the phosphopeptide flagged by the secondary run. In certain embodiments, the primary run and secondary run are hydrophobic separations based on chromatographic separation and the modification of the phosphopeptide is treatment with phosphatase. Dephosphorylated flagged peptides undergo a hydrophobic shift and can therefore be isolated from most unmodified peptides in each fraction by secondary runs on a hydrophobic column. It will be appreciated that specific phosphatases can be used to isolate specific phosphopeptides. To isolate a peptide containing phosphorylated tyrosine, a phosphatase specific for phosphotyrosine can be used.

  In yet another embodiment, the present invention relates to a method for isolating flagged peptides modified on methionine and / or cysteine from a protein peptide mixture, wherein: (a) a protein peptide mixture by primary run (B) isolating methionine flagged by secondary runs, (b) chemically modifying methionine and cysteine residues in peptides present in each fraction A method comprising the steps of: In yet another embodiment, the present invention provides a method for isolating a flagged peptide modified on cysteine from a protein-containing sample, wherein (a) oxidizing the protein sample; (b) ) Generating a protein peptide mixture, (c) separating the protein peptide mixture into fractions by a primary run, and (d) chemically modifying cysteine residues present in the peptides in each fraction. And (e) isolating cysteine-peptides flagged by secondary runs. In yet another embodiment, the present invention provides a method for isolating a flagged peptide modified on phospho-serine and / or phospho-threonine from a protein-containing sample, comprising: (a) a protein sample (B) separating the protein peptide mixture into fractions by primary run; (c) enzymatically modifying the peptide containing phosphoserine and / or phospho-threonine in each fraction; and (d) A method comprising isolating phospho-serine and phospho-trenion peptides flagged by a secondary run is provided.

  In yet another embodiment, the present invention provides a method for isolating a peptide subset from a protein peptide mixture, comprising: (a) separating the protein peptide mixture into fractions by a primary run; (b) each of the primary fractions. There is provided a method comprising the steps of adding a chelating agent to and (c) isolating the chelated peptide by a secondary run. The chelating compound can be a small complex-forming molecule, a cofactor, an antibody, and the like.

In yet another embodiment, the present invention provides a method for isolating a phosphorylated peptide from a protein peptide mixture, wherein (a) separating the protein peptide mixture into fractions by a primary run; (b) A method is provided comprising the step of adding at least one chelating compound to the primary fraction, and (c) isolating the phosphorylated peptide by a secondary run. In certain embodiments, the chelating compound used for the isolation of phosphorylated peptide comprises Fe ³⁺ and iminodiacetate.

  In yet another embodiment, the present invention provides a method for isolating a peptide subset from a protein peptide mixture, wherein (a) separating the protein peptide mixture into fractions by a primary run; (b) each fraction. A step of chemically or enzymatically adding a bulky and enormous entity to at least one amino acid in at least one of the peptides in the peptide, and (c) a peptide flagged from each fraction by secondary run The method includes the step of isolating, wherein the primary run and the secondary run are performed on the size exclusion column under identical or substantially similar conditions.

  In yet another embodiment, the invention provides a method for isolating a glycosylated peptide from a protein peptide mixture, comprising: (a) separating the protein peptide mixture into fractions by a primary run; b) chemically and / or enzymatically modifying glycosylation structures present on peptides within each fraction; and (c) isolating flagged peptides containing glycosylation structures modified by secondary runs. A method comprising the steps is provided. In certain embodiments, the modification of the glycosylation structure can be, for example, chemical and / or enzymatic deglycosylation, and alternatively, the glycosyl group is otherwise co-eluting. It can be converted into moieties with different biophysical or biochemical properties so that it can be separated from the modified peptide. For example, sialylated glycosyl chains can be desialylated by neuraminidase treatment, resulting in a shift on the chromatographic medium.

  In yet another embodiment, the present invention provides a method for isolating ε-N-acetylated peptides from a protein peptide mixture, comprising: (a) separating the protein peptide mixture into fractions by a primary run; Providing a method comprising the steps of: enzymatically deacetylating ε-N-acetylated peptides within each fraction; and (c) isolating deacetylated flagged peptides by secondary runs. is doing.

  As mentioned above, isolation of a subset of peptides flagged with the methods provided by the present invention requires that only a sub-population of peptides undergo modification within the protein-peptide mixture. In multiple fields of application, the modification can be performed directly on the peptide. However, (a) pretreatment of the protein in the sample and / or (b) pretreatment of the peptide in the protein peptide mixture allows to expand the range of peptide classes that can be isolated in the present invention. To illustrate the principle, an example of how cysteine-containing peptides can be isolated is described. It is clear that peptides containing one or more cysteines can be converted to more hydrophilic flagged peptides, for example by chemical reaction with acrylamide. This chemical modification converts cysteine to the more hydrophilic S-propionamide-cysteine. This cysteine derivative can be converted to an even more hydrophilic version by an oxidation reaction. Such oxidation converts S-propionamide-cysteine to S-propionamide-cysteine sulfoxide. Flagged peptides containing S-propionamide-cysteine-sulfoxide derivatives show a very significant hydrophilic shift and can therefore be easily isolated from the bulk of unmodified peptides using the present invention. However, by applying the chemical modifications described above, not only does the cysteine-containing peptide modify, but also the methionine-containing peptide (this also means that oxidation similarly converts methionine to its higher affinity methionine sulfoxide derivative. For conversion). As a result, two types of flagged peptides are produced simultaneously by modification and are also isolated simultaneously. In order to avoid such simultaneous isolation of Cys and Met peptides, a pretreatment step is introduced.

  In one particular embodiment, the protein in the mixture is oxidized before it is divided into its constituent peptides. This treatment results in the oxidation of methionine to its methionine sulfoxide derivative. The protein is then precipitated and reduced to convert disulfide bridges to thiol groups. The protein peptide mixture resulting from the protein splitting is then subjected to a primary run according to the present invention and the fraction is chemically modified with acrylamide and subsequently oxidized. Since methionine has already been oxidized during the pretreatment step, only the Cys-containing peptide is modified at this time. The flagged S-propionamide-cysteine sulfoxide peptide is isolated by applying a secondary run without significant Met-peptide contamination.

  This example illustrates that the selectivity of the modification reaction towards a selected amino acid (or modified amino acid or amino acid residue, etc.) can be obtained by pretreating the protein in the sample prior to the primary run. is doing. Such pretreatment can be directed equally well to the peptides in the protein peptide mixture prior to the primary run. In certain cases, pre-processing can also be performed during the primary run. Thereby, the present invention further provides a method for isolating flagged peptides from a protein peptide mixture in order to prevent co-modification of unwanted amino acids in a) step (c). (B) separating the protein peptide mixture into fractions by a primary run; (c) at least one amino acid in at least one peptide in each fraction; There is provided a method comprising the steps of chemically and / or enzymatically modifying and (d) isolating peptides flagged by secondary runs. Such pretreatment can include one or more chemical and / or enzymatic reactions.

In certain embodiments, flagged peptides derived from proteins with free NH ₂ termini can be isolated. The latter method includes the following steps: (a) pretreating a sample containing the protein to derivatize the cysteine side chain and convert lysine to homoarginine; (b) alpha-NH ₂ Converting the group into a thiocarbamoyl derivative; (c) preparing a protein peptide mixture; (d) blocking newly formed NH ₂ groups in the mixture; (e) treating the mixture with an acid. Inducing the loss of the NH ₂ terminal residue of the peptide blocked in step (d), (f) separating the pre-treated protein peptide mixture in the first chromatographic run, g) newly the generated NH ₂ group modified with acetylated compound process for generating a subset of peptides erected whereby the flag; and (h) secondary chromatography Isolating the peptide stood the flag Rafi process. In certain cases, step d) of the latter embodiment can be carried out with trinitrobenzenesulfonic acid (TNBS). In another particular case, step e) is performed with TFA.

  As described above, in the reverse action mode, the present invention provides a method for isolating an identified peptide from a protein peptide mixture. In this embodiment, a small number of peptides in the protein peptide mixture remain unmodified, while the bulk of the peptide remains modified. The modified peptide acquires properties that alter its chromatographic behavior, while the identified peptide remains unaltered and retains its original chromatographic behavior. Thus, the identified peptides elute simultaneously during the secondary run as was done during their primary run, while the modified peptide is shifted back and forth. In this way, it is possible to separate the identified peptide from the modified peptide and isolate the identified peptide in each fraction.

In a manner similar to the situation with flagged peptides as described above, the present invention requires that the modification be effective in each of the peptide fractions from the primary run. Thereby, within each fraction obtained from the primary chromatography step, the modified peptide must migrate quite differently from the identified peptide in the secondary chromatography step. Depending on the type of modification applied, the shift can be caused, for example, by changes in hydrophobic hydrophilicity or net charge. This shift is called δp and is specific for all individual modified peptides. In examples of hydrophobic changes, the δp value can be expressed as a change in hydrophobic moment or as a percentage of organic solvent in a chromatographic run, but most practically, for a given chromatographic / electrical It can be expressed in units of time under electrophoresis conditions. Thereby, δp is not necessarily the same for all modified peptides and exists between δmax and δmin. δp is affected by a certain number of factors such as the nature of the induced modification, the nature of the column stationary phase, the mobile phase (buffer, solvent), temperature, etc. In the example where peptides in the fraction from the primary run elute within window w ₁ , the identified peptide will elute within approximately the same window w ₁ during the secondary run. In a preferred embodiment, δmin must be sufficient to avoid the modified peptide eluting within window w ₁ (and thus overlapping with the identified peptide). This law should be applied to all fractions collected from the primary run. Preferentially, δmin should be w ₁ or higher to minimize the overlap between the modified peptide and the identified peptide. For example, if w ₁ = 1 minute, δmin should be greater than 1 minute if possible. However, it is self-evident that slight contamination of the modified peptide within the elution window of the identified peptide is undesirable but acceptable. Manipulation of the value of δmin to obtain an optimal separation of the identified peptides from the modified peptides within each primary run fraction is part of the present invention, among others selected to be in a modified state. The correct combination of amino acid (s), modification type and chromatographic conditions (column type, buffer, solvent, etc.). It will be apparent to those skilled in the art that the same approach can be applied to isolate identified peptides, for example, by ion exchange chromatography or other types of chromatography.

  Accordingly, the present invention further provides a method for isolating a peptide subset from a protein peptide mixture, the step of (a) separating the protein peptide mixture into peptide fractions by chromatography; (b) at least 50%, preferably 60, within each fraction. %, More preferably 70%, even more preferably 80%, and most preferably 90% or more of the peptide chemically and / or enzymatically modified; and (c) isolating the identified peptide by chromatography Including, wherein the chromatography of steps (a) and (c) is carried out with the same type of chromatography. Similar to the flagged peptide approach, alterations between primary and secondary runs include, for example, amino acids, modified amino acids (glycosylation, phosphorylation, acetylation, etc.), certain amino acids in vitro Modifications and / or alterations of specific residues on one or more amino acids.

  As mentioned above, isolation of a subset of identified peptides with the methods provided by the present invention requires that most peptides be modified within the protein peptide mixture. In multiple fields of application, this modification can be performed directly on the peptide. However, (a) pretreatment of the protein in the sample and / or (b) pretreatment of the peptide in the protein peptide mixture allows to expand the range of peptide classes that can be isolated in the present invention. In certain cases, pre-processing can also be performed during the primary run.

To illustrate the principle, we describe an example of how an amino terminal blocked peptide (ie, a peptide derived from the amino terminus of a protein blocked in vivo at its amino terminus) can be isolated. Amino-terminally blocked peptides are in accordance with the present invention (a) separating the protein peptide mixture by chromatography; (b) modifying the free amino terminal group of the peptide (derived from the protein blocked at the amino terminus). And (c) separating the identified peptide blocked at the amino terminus from the bulk of the peptide modified by chromatography (where steps (a) and (e) ) Chromatography can be performed by the same type of chromatography). Although this approach makes it possible to isolate many of the amino-terminally blocked peptides, no population of amino-terminally blocked peptides containing lysine is selected in the procedure. Lysine also has a free amino group, so modification in step (b) will also modify peptides blocked at the amino terminus containing lysine as well. The population of peptides blocked at the amino terminus, which also contains lysine, is thereby modified and thus does not form part of the identified peptide and will not be isolated. To avoid this loss of population, the protein lysine prior primary run epsilon-NH ₂ - pretreatment step to convert the amino group blocked group is introduced. In certain embodiments, lysine with a free ε-NH ₂ -group is converted to homoarginine followed by digestion with trypsin, which splits the protein in homoargin and free α-amino acids are Generated at these positions. As a result, the amino-terminal blocked peptide containing lysine is no longer modified and is isolated as an identified peptide.

  This example shows that pre-treatment of the protein in the sample prior to the primary run enhances the selectivity of the modification reaction towards the selected amino acid (or modified amino acid or amino acid residue). Yes. Such pretreatment is equally well directed to peptides within a protein peptide mixture. The invention further thereby provides a method for isolating an identified peptide from a protein peptide mixture: a) pretreatment of the peptide in the protein peptide mixture and / or protein in the sample; (b) fractionating the protein peptide mixture by a primary run (C) chemically and / or enzymatically modifying at least one amino acid in most of the peptides in each fraction; and (d) isolating the identified peptide by a secondary run. Provides a way to include. Such pretreatment can include one or more chemical and / or enzymatic reactions.

The present invention further relates to a method for isolating a peptide blocked at the amino terminus of a protein in a protein-containing sample, wherein (1) the protein lysine ε-NH ₂ — group is converted to a guanidyl group and other moieties. (2) digesting the protein sample in such a way that the protein is split in homoarginine to produce free α-amino acids at these positions; (3) fractionating the protein peptide mixture within the primary run; 4) providing a method comprising the step of modifying the free amino terminal group of the peptide in each fraction with a hydrophobic, hydrophilic or charged component and (5) isolating the unmodified identified peptide in a secondary run. .

In yet another embodiment, the present invention provides a method for isolating the amino terminal peptide of a protein in a sample containing the protein. This method comprises the following steps: (1) converting the protein lysine ε-NH ₂ group to a guanidyl group or other moiety; (2) converting a free α-amino group on the amino terminal side of each protein. Generating a blocked (non-reactive) group; (3) digesting a protein sample to produce a peptide with newly generated free NH ₂ groups; (4) a protein peptide mixture in the primary run (5) modifying the free NH ₂ groups of peptides in each fraction with hydrophobic, hydrophilic or charged components, and (6) isolating unmodified identified peptides with secondary runs. This approach involves specifically isolating the amino terminal peptide of a protein in a protein sample, including both an amino terminal peptide with a free α-amino acid group and an amino terminal peptide with a blocked α-amino acid group. enable. Block in vivo with an amino-terminal peptide having a free NH ₂ group by performing step 2 in the above protocol in such a way that the free α-amino group is blocked with an isotopically labeled residue. It is possible to identify the amino terminal peptide that has been made. One area of application of the latter embodiment is the study of internal proteolytic processing of proteins between two different samples containing proteins (see, eg, Example 8).

  In yet another embodiment of the reverse action mode, chemical or enzymatic modification between the primary run and the secondary run is present in most peptides. Targeted to amino acids. Such abundant amino acids are present in at least 50% peptide, preferably more than 75% peptide, more preferably more than 90% peptide.

  In another embodiment, the identifying peptide is the COOH-terminal (carboxy-terminal) peptide of the protein.

  In yet another embodiment, the present invention provides a method for isolating a peptide subset from a protein peptide mixture, wherein (a) separating the protein peptide mixture into fractions by a primary run; (b) each fraction. Chemically or enzymatically adding a bulky and enormous entity to at least one amino acid in most of the peptides, and (c) isolating said identified peptide from each fraction by secondary run And a method wherein the primary run and the secondary run are performed on the size exclusion column under identical or substantially similar conditions.

  The method according to the invention makes it possible to separate flagged peptides from most unmodified peptides within each fraction, and finally a specific subset of flagged peptides from the complete protein peptide mixture. Result in isolation. As noted above, such flagged peptides include, for example, peptides containing one or more methionines, peptides containing one or more cysteines, peptides containing one or more methionines and / or one or more cysteines, phospho It may be a peptide, a peptide phosphorylated on tyrosine, a peptide containing ε-N-acetylated cysteine. Flagged peptides represent high levels of derived proteins, so flagged peptides serve as identification elements for their corresponding proteins. Accordingly, the present invention further provides a method for identifying peptide subsets and their corresponding proteins in a sample containing proteins. Moreover, flagged peptide isolation according to any of the embodiments of the present invention is further coupled with peptide analysis.

  Similarly, the method according to the invention allows separation of the identified peptides from the majority of the modified peptides within each fraction, and finally the specific subset of identified peptides from the complete protein peptide mixture. Result in isolation. As described above, such identification peptides can be, for example, amino acid terminal peptides, amino acid blocked peptides, carboxy terminal peptides. Identification peptides represent high levels of derived proteins, so identification peptides serve as identification elements for their corresponding proteins. Thus, the present invention further provides a method for identifying peptide subsets and corresponding proteins in a sample containing proteins. Moreover, flagged peptide isolation according to any of the embodiments of the present invention is further coupled with peptide analysis.

  In a preferred approach, peptide analysis of flagged or identified peptides is performed on a mass spectrometer. However, flagged or identified peptides can also be further analyzed and identified using other methods such as electrophoresis, activity measurement in assays, analysis with specific antibodies, Edman sequencing, and the like.

  The analysis and identification steps can be performed in different ways. In one way, the flagged or identified peptide eluting from the chromatography column is directed directly to the analyzer. In an alternative approach, flagged or identified peptides are recovered in the form of fractions. Such a fraction may or may not be manipulated before proceeding to further analysis or identification. An example of such an operation consists of a concentration step followed by spotting of each concentrate on, for example, a MALDI-target for further analysis and identification.

  In preferred embodiments, flagged or identified peptides are analyzed with high-throughput mass spectrometry techniques. The information obtained is the mass of the flagged or identified peptide. Once a peptide mass has been defined very accurately on a Fourier Transform Mass Spectrometer (FTMS) using, for example, an internal calibration procedure (O'Connor and Costello, 2000), the peptide mass is matched to the corresponding peptide mass database. It is possible to identify peptides that are unambiguously correlated with the mass of the peptide and thereby flagged or identified. However, the accuracy of some conventional mass spectrometers is not sufficient to unambiguously correlate the spectroscopically determined mass of each peptide with its corresponding peptide and protein in the sequence database. In order to increase the number of peptides that can still be clearly identified, the data about the mass of the peptide is supplemented with other information. In one embodiment, the mass of the peptide as determined by the mass spectrometer is proven knowledge that each flagged peptide contains one or more residues of modified amino acids (eg, In the case of peptides with a methionine sulfoxide flag, this is evidenced by a neutral loss of 64 amu) and / or the peptide was generated after digestion of a sample containing the protein using a split protease with known specificity It is complemented with the knowledge. For example, trypsin has the well-known property of splitting precisely at the lysine and arginine sites to produce peptides with a C-terminal lysine or arginine amino acid typically having a molecular weight of about 500-5000 daltons. This combined information is used to screen a database containing information regarding peptide mass, sequence and / or identity and to identify corresponding peptides and proteins.

In another embodiment, the method of determining the identity of a parent protein by only accurately measuring the peptide mass of at least one flagged peptide or an identified peptide comprises a selected flagged peptide Alternatively, it can be improved by further enriching the information content of the identified peptide. As a non-limiting example of how information can be added to flagged or identified peptides, the free NH ₂ groups of these peptides are added with two different isotope-labeled groups. Can be chemically altered specifically in the chemical reaction. As a result of this change, the peptide acquires a predetermined number of labeled groups. Because a modified agent is a mixture of two chemically identical but isotopically different agents, flagged or identified peptides are revealed as peptide pairs in the mass spectrum. The The range of mass shift between these peptide doublets represents the number of free amino acids present in the peptide. To further illustrate this, for example, the information content of the peptides flagged, specifically the free NH ₂ groups in the peptides using an equimolar mixture of acetic acid N- hydroxysuccinimide ester and Torideutero acetate N- hydroxysuccinimide ester Can be enriched by changing As a result of this conversion reaction, the peptide acquires a predetermined number of CH ₃ —CO (CD ₃ —CO) groups so that it can be easily deduced from the range of mass shifts observed in peptide doublets. Thus, 3 Am correspond shift (amu) and one NH ₂ group, 3 and the shift of 6amu corresponds with two NH ₂ groups, 3, 6, and the shift of 9amu three NH within the peptide ₂ Reveal the presence of the group. This information further supplements data on peptide mass, knowledge about the presence of one or more residues of the modified amino acid, and / or knowledge that the peptide was generated using a protease with a known specificity.

  Yet another information element that can be used to identify flagged or identified peptides is the hydrophobic hydrophilicity of the peptide (GRAVY) as reflected in the elution time during chromatography. Two or more peptides with the same mass or mass that falls within the error range of mass measurement can be identified by comparing their experimentally determined GRAVY with the GRAVY predicted in the computer.

  Any mass spectrometer can be used to analyze the flagged or identified peptides. Non-limiting examples of mass spectrometers include matrix-assisted laser desorption / ionization ("MALDI" time-of-flight ("TOF") mass spectrometry MS or MALDI- available from Perseptive Biosystems, Framingham, Massachusetts TOF-MS, Reflex III from Brucker-Daltonias, Bremen, Germany and Ettan MALDI-TOF from AP Biotech; Electrospray ionization (ESI available from Finnigan MAT, San Jose, California ) Ion trap mass spectrometer; ESI quadrupole mass spectrometer available from Finnigan MAT or GSTAR pulser hybrid LC / MS / MS system from Applied BioSystems Group, Foster City California, and Fourier transform mass using internal calibration procedure An analyzer (FTMS) (O'Connor and Costells, 2000) is included.

  Protein identification software used in the present invention to compare peptide experimental mass spectra with peptide masses and corresponding protein databases is available in the art. One such algorithm, Pro Found, uses a Bayesian algorithm to search for proteins and a DNA database to identify the best match between experimental data and proteins in the database. ProFound can be accessed on the World-Wide web at <http // prowl.rockefeller.edu> and <http // www.proteometricks.com>. ProFound accesses a non-redundant database (NR). Peptide searches are accessible on the EMBL website. Similarly, Chaurand P. et al. (1999) J. Am. Soc. Mass. Spectrom 10, 91, Patterson SD, (2000), Am. Physiol. Soc. 59-65, Yates JR (1998) Electrophoresis, 19 See also 893. MS / MS spectra can also be analyzed by MASCOT (available from Matrix Science Ltd. London at http://www.matrixscience.com.).

In another preferred embodiment, the isolated flagged or identified peptide is individually subjected to fragmentation in a mass spectrometer. In this way, information regarding the mass of the peptide is further supplemented with (partial) sequence data for the flagged or identified peptide. By comparing this combined information with information in the peptide mass and peptide and protein sequence databases, it is possible to identify flagged or identified peptides. In one approach, fragmentation of flagged or identified peptides is most conveniently performed by collision-induced dissociation (CID), commonly referred to as MS ² or tandem mass spectrometer. Alternatively, flagged or identified peptide ions may decay during their flight after volatilization and ionization in MALDI-TOF-MS. This process is called post-source decomposition (PSD). In one such mass spectrometry approach, a selected flagged or identified peptide is transferred directly or indirectly into the ion source of an electrospray mass spectrometer and then in MS / MS mode. It is further fragmented. Thereby, in one embodiment, partial sequence information of the flagged peptide or identified peptide is recovered from the MS ⁿ fragment spectrum (where n is greater than or equal to 2) and within the sequence database described herein. Used for peptide identification.

In certain embodiments, additional sequence information can be obtained in a MALDI-post source degradation analysis when the alpha-NH ₂ -terminus of the peptide is modified with a sulfonic acid moiety. Peptides flagged with a sulfonic acid group at the NH ₂ terminus are induced according to a specific fragmentation pattern when detected in MALDI-TOF-MS mode. This mode allows for quick and easy deduction of amino acid sequences. In particular, example 6b is, NH ₂ - peptides made a terminal flag describes whether procedures are isolated by how the proteins with a free NH ₂ -terminal.

  The invention further provides a method for the identification of one or more proteins in a sample containing proteins. On the one hand, it is known that splitting a sample containing protein results in a protein peptide mixture containing thousands of peptides, which exceeds the resolution capabilities of currently available chromatography and mass spectrometry systems. . On the other hand, it has been found that a protein can be identified based on the identification of one or more of its constituent peptides. The present invention provides a method for isolating and identifying a wide variety of different types of flagged or identified peptides from a protein peptide mixture. All sets of flagged or identified peptides represent one subset of peptides within a protein peptide mixture. This simplification of the original peptide mixture significantly reduces the co-elution of peptides in the secondary run, resulting in efficient identification of peptides flagged or identified in analyzers such as mass spectrometers and others Bring as. Since a flagged or identified peptide is most often a unique identifying element for its corresponding parent protein, identification of the flagged or identified peptide will result in the original sample containing the protein. Protein identification becomes possible. Thus, the task of identifying proteins in a sample containing proteins by isolating and identifying one or more of the complex peptides is possible using the methods of the present invention.

  Accordingly, the present invention further provides a method for identifying a protein in a sample containing protein, wherein (a) separating the protein peptide mixture into chromatographic fractions by chromatography; (b) of the peptides in each fraction Chemically and / or enzymatically modifying at least one amino acid of at least one peptide to produce a subset of modified peptides, and (c) peptides flagged from each fraction by secondary runs Isolating; (d) identifying the flagged peptide and its corresponding protein.

  Accordingly, the present invention further provides a method for identifying a protein in a sample containing protein, wherein (a) separating the protein peptide mixture into peptide fractions by chromatography; (b) the majority of peptides within each fraction; Chemically and / or enzymatically modifying at least one of the amino acids to produce a subset of unmodified peptides, and (c) isolating the identified peptide from each fraction by secondary run; (d) There is also provided a method comprising identifying an identifying peptide and its corresponding protein.

  For those skilled in the art, starting from the known identity of flagged or identified peptides, it is easy to determine the identity of the corresponding protein by screening peptide, protein and DNA sequence databases. It is equally obvious. Both software and databases for screening are available in the art.

  Flagged peptides that can be used in accordance with the present invention to identify proteins in samples containing proteins include, for example, methionine containing peptides, cysteine containing peptides, histidine containing peptides, tyrosine containing peptides, lysine containing peptides, tryptophan containing peptides Arginine-containing peptides, proline-containing peptides, phenylalanine-containing peptides, or combinations of two or more of these flagged peptides.

  Other flagged peptides can be used in accordance with the present invention to identify the presence of a protein that has been modified simultaneously or post-translation in a sample containing the protein. For example, the present invention provides a method for identifying a phosphorylated protein in a sample containing the protein. Thus, in one approach, a peptide containing a phosphorylated amino acid is modified according to the present invention and isolated as a flagged peptide. Subsequent identification of these flagged peptides and their associated proteins results in the identification of phosphorylated proteins (or phosphoproteomes) in the sample. The present invention also applies to others in samples containing proteins such as glycosylated proteins, tyrosine-phosphorylated proteins, serine- and / or threonine phosphorylated proteins, acetylated proteins, ε-N-acetylated proteins, sulfated proteins, etc. Also provided are methods for identifying proteins that have been modified simultaneously or post-translationally.

  An identifying peptide that can be used in accordance with the present invention to identify a protein in a sample is, for example, the amino terminal peptide of the protein. The mass of each of these peptides can be determined using mass spectrometry. Combining the mass of such peptides with the knowledge that such peptides are amino-terminal peptides is sufficient to unambiguously identify the corresponding parent peptide for most peptides. In a further embodiment of this aspect of the invention, databases containing only the amino terminal peptide masses are designed, against which the mass of the isolated amino terminal identifying peptide is probed. With this approach, there is a very high probability that the isolated identified peptide will uniquely match the mass in the limited database. In addition, this approach significantly reduces the complexity of the peptide-based proteome approach and significantly increases the analysis speed.

  That the present invention allows the identification of the entire range of proteins in a sample, including proteins that vary from acidic to basic, from small to large, from soluble to membrane proteins, eg, from high to low abundance It is also important to mention. Furthermore, the present invention provides a method for identifying proteins in a sample containing proteins starting from a very small amount of cells. The method provided by the present invention is very efficient and sensitive so that, for example, hundreds to a thousand or more proteins can be identified starting from as few as 50,000 human cells. Is possible. Even if fewer cells are used as starting material, it is still possible to identify hundreds of proteins in a sample containing proteins. Obviously, the method of the invention can be applied to a large number of cells as well. Other identifying peptides can be used, for example, to identify proteins blocked at the amino terminus or proteins that have been cleaved by proteolysis.

  In another embodiment, the present invention provides a method for determining the relative amount of one or more proteins in two or more samples containing proteins. This method involves the use of differentially isotopically flagged or identified peptides. In this method, two samples are flagged peptides or identified peptides isolated from one sample and flagged peptides or identified peptides isolated from the second sample contain one isotope. Is processed in such a way that it contains another isotope of the same element.

  The method includes (a) labeling a peptide present in a first sample with a first isotope; (b) a peptide present in a second sample with a second isotope. Labeling; (c) combining the protein peptide mixture of the second sample and the protein peptide mixture of the first sample; (d) separating the protein peptide mixture into a peptide fraction by chromatography; e) chemically or enzymatically or chemically and enzymatically modifying at least one amino acid of at least one of the peptides in each fraction; and (f) flagging each fraction by chromatography. Isolating the erected peptide, the chromatography being the same as in step (d) (G) performing a mass spectrometric analysis of the isolated flagged peptide; (h) a flag that is identical but differentially labeled with an isotope. Calculating the relative amount of flagged peptides in each sample by comparing the peak heights of the flagged peptides; and (i) determining the identity of the flagged peptide and its corresponding protein. Process is included.

  The same approach can be followed with reverse mode action, in which case the method comprises (a) labeling the peptide present in the first sample with the first isotope; Labeling peptides present in the second sample with two isotopes; (c) mixing the protein peptide mixture of the second sample with the protein peptide mixture of the first sample; (d) the protein peptide Separating the mixture into fractions of peptides by chromatography; (e) chemically or enzymatically or chemically and enzymatically modifying at least one amino acid of most of the peptides in each fraction And (f) isolating the identified peptide from each fraction by chromatography, A step in which chromatography is carried out with the same type of chromatography as in step (d); (g) a mass spectrometric analysis of the isolated identified peptide; (h) an identical but differential isotope. Calculating the relative amount of the identified peptide in each sample by comparing the peak heights of the identified peptide labeled with, and (i) determining the identity of the identified peptide and its corresponding protein. include.

  As mentioned above, it is clear that the same approach can be used in combination with a pretreatment step. The method is also applicable when the chromatographic separations in steps (d) and (f) are identical or substantially similar. Similarly, instead of mixing the peptides from both samples within step (c), the peptides from the first and second samples are separately separated from steps (d) and / or (e) and / or (f). It is also clear that the combined state in step (d) or (e) of (f) or (g) can be obtained.

Differential isotope labeling of peptides in the first and second samples can be performed in a number of different ways available in the art. The key element is that a particular peptide derived from the same protein in the first and second samples is identical except that there are different isotopes within one or more amino acids of the peptide. . In one standard embodiment, the isotope in the first sample is a natural isotope, meaning an isotope that is widely present in nature, and the isotope in the second sample is the rare isotope below. This is a less common isotope. Examples of natural and rare isotope pairs are H and D, O ¹⁶ and O ¹⁸ , C ¹² and C ¹³ , N ¹⁴ and N ¹⁵ . In the present application, a peptide labeled with a heavier isotope of an isotope pair is also called a heavy peptide. Peptides labeled with the lighter isotope of an isotope pair are also referred to herein as light peptides. For example, a peptide labeled with H is called a light peptide, while the same peptide labeled with D is called a heavy peptide. Peptides labeled with natural isotopes and their counterparts labeled with rare isotopes are chemically very similar and are chromatographically separated and ionized in the same manner. However, when supplied into an analyzer such as a mass spectrometer, the peptides are separated into light and heavy peptides. The heavy peptide has a slightly higher mass due to the greater weight of the selected isotope label incorporated. Since the difference between the masses of the differentially isotopically labeled peptides is small, as a result of mass analysis of the isolated flagged peptide or the identified peptide, dense peptides representing heavy and light peptides, respectively. Results in multiple pairs of two peaks that are spaced apart. Each heavy peptide is derived from a sample labeled with a heavy isotope, and each light peptide is derived from a sample labeled with a photoisotope. The ratio (relative abundance) of the peak intensity of the heavy and light peaks in each pair is then measured. These ratios provide a measure of the relative amount (differential incidence) of that peptide (and its corresponding protein) within each sample. The peak intensity can be calculated in a conventional manner (eg, by calculating peak height or peak surface area). As described herein, flagged or identified peptides can also be identified to allow identification of proteins in a sample. If a protein is present in one sample but not in another sample, the isolated flagged or identified peptide (corresponding to this protein) is either heavy or light isotope It will be detected as one peak that may contain. However, in some cases, it can be difficult to determine which sample produced a single peak observed during mass analysis of a combined sample. This problem can be solved by double labeling the first sample with two different isotopes or with two different numbers of heavy isotopes before or after proteolytic resolution. An example of a labeling agent is an acylating agent.

  Incorporation of natural and / or rare isotopes within the peptide can be obtained in a number of ways. In one approach, the protein is labeled intracellularly. Cells for the first sample were grown, for example, in medium supplemented with amino acids containing natural isotopes, and cells for the second sample were supplemented with amino acids containing rare isotopes. Grown in medium. In one embodiment, the differentially isotopically labeled amino acid is an amino acid selected to be in a modified state. For example, if methionine is the selected amino acid, the cells are either unlabeled L-methionine (first sample) or deuterated at the Cβ and Cγ positions and thus heavier by 4 amu. In the supplemented medium.

  The mixing of proteins / peptides from both samples can be done at different times. Mixing can be done at the sample level (eg, mixing equal numbers of cells from both samples), or alternatively, proteins can be isolated from Sample 1 and Sample 2 separately and then mixed. It is also possible to digest the protein from sample 1 into a peptide, digest the protein from sample 2 into a peptide, mix the peptides from sample 1 and sample 2, and so on. Regardless of the mixing procedure, the present invention is further used to isolate flagged methionine-peptides from protein peptide mixtures. The methionine-peptide will be isolated regardless of its isotopic composition, and analysis of the methionine peptide in the mass spectrometer as described above will result in the relative amount of its corresponding protein in sample 1 and sample 2 Can be determined.

Different isotope incorporations can also be obtained by enzymatic approaches. For example, one sample containing protein is treated with trypsin in “normal” water (H ₂ ¹⁶ O), and a sample containing a _second protein is treated with trypsin in “heavy” water (H ₂ ¹⁸ O). By doing so, labeling can be performed. “Heavy water” as used herein refers to a water molecule in which the O atom is an ¹⁸ O-isotope. Trypsin exhibits the well-known property of incorporating two hydrogens of water at the COOH-terminus of the newly generated site. Thereby, in sample 1 trypsinized in H ₂ ¹⁶ O, the peptide has a “normal” mass, whereas in sample 2 the peptide (except for most COOH-terminal peptides) contains two ^It has a mass increase of 4 amu corresponding to the uptake of ¹⁸ O atoms. This 4 amu difference is sufficient to identify heavy and light versions of the flagged or identified peptides in the mass spectrometer and thereby determine the corresponding peptide / protein ratio in the two samples. Thus, the present invention further provides a method for determining the relative amount of at least one protein in at least two samples, a) digesting the first sample protein with trypsin in the presence of H ₂ ¹⁶ O. Digesting a second sample protein with trypsin in the presence of H ₂ ¹⁸ O; b) combining two trypsin digested protein peptide mixtures; c) subjecting the combined mixture to a primary run ( Differentially isotopically labeled peptides have the same chromatographic behavior and therefore separate into the same fractions); d) chemically and / or at least one amino acid of at least one peptide within each fraction A step of enzymatic modification; e) a peptide flagged by a secondary run or the same Isolating peptides (differentially labeled flagged peptides or identified peptides are sorted within the same fraction since they have the same chromatographic behavior); f) in a mass spectrometer Analyzing released peptides; g) calculating the relative amount of corresponding heavy and light peptides by including their peak heights; and h) identifying the peptides and their corresponding proteins. Providing a way.

Differential isotope incorporation can also be obtained with a number of labeling procedures based on known chemical reactions that can be performed at the protein or peptide level. For example, the protein produces homoarginine lysine position preceding each thereby to convert the NH ₂ group in guanidinium group can be varied by Guajiniru reaction with O- methylisourea. Proteins from the first sample can be reacted with reagents with natural isotopes, and proteins from the second sample can be reacted with reagents with rare isotopes. The peptides could also be modified by Schiff base formation with deuterated acetaldehyde followed by reduction with normal or deuterated sodium borohydride. This reaction, known to proceed under mild conditions, can lead to the incorporation of a predictable number of deuterium atoms. The peptide will be modified either at the α-NH ₂ -group or the ε-NH ₂ group of lysine or both. Similar changes can be made with deuterated formaldehyde, followed by reduction with deuterated NaBD ₄ , which will produce a methylated version of the amino group. The reaction with formaldehyde is carried out on the whole protein with deuterium incorporated only into the lysine side chain or on a peptide mixture that will be labeled with both α-NH ₂ and NH ₂ groups from lysine. be able to. Since arginine does not react, this also provides a way to make a distinction between Arg-containing peptides and Lys-containing peptides. Primary amino groups are readily acylated, for example with acetyl N-hydroxysuccinimide (ANHS). Thereby, one sample can be acetylated with normal ANHS, while the second sample can be acylated with either ¹³ CH ₃ CO—NHS or CD ₃ CO—NHS. All lysine ε-NH ₂ groups are derivatized in this manner in addition to the amino terminus of the peptide. Still other labeling methods include trimethylchlorosilane, which can be used for less specific labeling of hydroxyl and amine containing functional groups, and anhydrous anhydrides that can be used to acetylate hydroxyl groups. There is acetic acid.

  In yet another approach, the primary amino acids are labeled with chemical groups that allow the heavy and light peptides to be distinguished by 5amu, 6amu, 7amu, 8amu and even greater mass differences. Examples of such compounds are mentioned in Example 16. Alternatively, differential isotope labeling is performed at the carboxy terminus of the peptide, allowing heavy and light variants to be distinguished by a mass difference of 5 amu or more, 6 amu, 7 amu, 8 amu or more and even greater. The method of the present invention does not require prior knowledge of the protein types that may be present in the sample and is therefore used to determine the relative amounts of both known and unknown proteins present in the sample under test. be able to.

  The methods provided in the present invention for determining the relative amount of at least one protein in at least two samples include cells, tissues or biological fluids (eg, nipple aspirate, saliva, semen, cerebrospinal fluid, urine, (Sera, plasma, synovial fluid), organs and / or can be widely applied to compare protein levels in the whole organism. Such comparisons include, for example, pathological and non-pathological, stressed and unstressed, drug-treated and not, benign and malignant, sticky and non-sticky, infected and uninfected Incorporated, subcellular fractions, transformed and untransformed, cells, tissues, body fluids, organs and / or whole assessments. This method can also be applied to different stimuli or in different developmental processes, or one or more genes are precipitating or overexpressed, or one or more genes are knocked out It is also possible to compare protein levels in subcellular fractions, cells, tissues, body fluids, living organisms, and whole organisms.

  In another embodiment, the methods described herein are similarly (eg, cancer, neurodegenerative disease, inflammation, cardiovascular disease, viral infection, bacterial infection, fungal infection or any other disease). It can also be used in diagnostic assays for the detection of the presence or absence of expression of specific protein sets or one or more protein markers that are indicative of a pathological condition. Specific fields of use include identification of target proteins present in metastatic and invasive cancers, differential expression of proteins in transgenic mice, identification of up- or down-regulated proteins in diseased tissues, metabolism Identification of intracellular changes in cells with physiological changes such as shifts, identification of biomarkers in cancer, identification of signaling pathways.

  Quantitative analysis of large protein sets in different samples can be performed using both flagged and identified peptides. In a standard example, a flagged peptide based on modification of methionine or cysteine or histidine or a combination of two of these amino acids will be used. In another standard example, identification peptides based on amino-terminal or carboxy-terminal peptides are used. Furthermore, the present invention can be used to achieve qualitative and quantitative analysis of the entire proteome of the protein modification state. For example, in multiple signal translation pathways, serine, threonine, and tyrosine residues present in proteins are often phosphorylated. In one specific embodiment, the differentially isotopically flagged peptide is a flagged peptide selected based on the presence of phospho-amino acids. Comparison of the relative abundance of peptides that are flagged heavy and light makes it possible to compare the relative abundance of phosphorylated proteins in two samples containing proteins. In yet another embodiment, the differentially isotopically labeled flagged peptide is a flagged peptide selected based on the presence of phosphoserine and / or phosphothreonine or phosphotyrosine. In yet another embodiment, the differentially isotope-labeled flagged peptide is a flagged peptide selected based on the presence of an ε-N-acetylated lysine-containing peptide. In yet another embodiment, the differentially isotopically flagged peptide is a flagged peptide selected based on the presence of a glycosyl group.

The invention further provides a method for quantifying the amount of one or more proteins in a single sample containing the protein. This method
(A) preparing a protein-peptide mixture; (b) adding a known amount of a synthetic reference peptide labeled with an isotope distinguishable from a reference peptide isotope to the mixture, whereby said synthetic reference peptide and said A step wherein the reference peptide counterpart is to be modified in step (d); (c) separating the mixture into peptide fractions by chromatography; (d) of at least one of the peptides in each fraction; Chemically or enzymatically or chemically and enzymatically modifying at least one amino acid; and (e) isolating flagged peptides from each fraction by chromatography comprising the steps of: Wherein is carried out in the same type of chromatography as in step (c); Comprising the step of determining the amount of protein present in the sample by comparison with (g) a reference peptide peak heights of the synthetic reference peptides;) carrying out the analysis by mass spectrometry of the peptides flagged process.

  The same method can be applied in reverse mode action, where the method comprises: (a) preparing a protein peptide mixture; (b) a synthetic standard labeled with an isotope distinguishable from a reference peptide isotope. Adding a known amount of peptide to the mixture, whereby the synthetic reference peptide and the reference peptide counterpart will be modified in step (d); (c) separating the mixture into peptide fractions by chromatography (D) chemically or enzymatically or chemically and enzymatically modifying at least one amino acid of most of the peptides in each fraction; and (e) each fraction by chromatography. Isolating the identified peptide from, wherein the chromatography is the same as in step (c) (F) performing a mass spectrometric analysis of the identified peptide; (g) a protein present in the sample by comparing the peak height of the synthetic reference peptide with the reference peptide Calculating the amount of.

  As mentioned above, it is clear that the same method can be used in combination with a pretreatment step. The method is also applicable when the chromatographic separation in steps (c) and (e) is the same or substantially similar.

  A “reference peptide” is herein a peptide having sufficient sequence and / or mass to unambiguously identify its parent protein. Peptide synthesis equivalent to the reference peptide is preferably easy. For clarity, the reference peptide used in this application is the native peptide as found in the protein it represents, while the synthetic reference peptide used in this application is the synthesis of the same peptide. It is a counterpart. Such synthetic reference peptides are suitably produced by peptide synthesis, but can also be produced recombinantly. Recombinant production can be obtained in a number of vectors and hosts, as is widely available in the art. The reference peptide is preferably fully ionized in mass spectrometry. A non-limiting example of a reference peptide that fully ionizes is a reference peptide containing arginine. Preferably, the reference peptide can also be readily isolated as a flagged peptide or an identified peptide. In the latter preferred embodiment, the reference peptide is also a flagged peptide or an identifying peptide.

  The reference peptide and its synthetic reference peptide counterpart are chemically very similar and are separated in the same manner by chromatography and ionized in the same manner. However, the reference peptide and its synthetic reference peptide counterpart are differentially labeled with isotopes. Thus, in a preferred embodiment where the reference peptide is also a flagged peptide or an identification peptide, the reference peptide and its synthetic reference peptide counterparts are modified in a similar manner, primary and secondary runs and possibly Isolated in the same fraction of the tertiary run. However, the reference peptide and its synthetic reference peptide are separated into light and heavy peptides when supplied to an analyzer such as a mass spectrometer. The heavy peptide has a slightly higher mass due to the greater weight of the selected heavy isotope incorporated.

  Because of this very small mass difference between the reference peptide and its synthetic reference peptide, both peptides will appear as two closely spaced peaks that are recognizable in mass spectrometry. The ratio between peak height or peak intensity can be calculated, which determines the ratio of the amount of reference peptide to the amount of synthetic reference peptide. Since a known absolute amount of a synthetic reference peptide is added to the protein peptide mixture, the amount of the reference peptide can be easily calculated and the amount of the corresponding protein in the sample containing the protein can be calculated .

Several methods are known in the art for differentially isotopically labeling a reference peptide and its synthetic reference peptide. In the first approach, the reference peptide carries rare isotopes and the synthetic counterpart carries natural isotopes. In this approach, a synthetic reference peptide can be chemically efficiently synthesized with its natural isotopes in large-scale preparations and a synthetic reference peptide. To label a reference peptide with a rare isotope, any of the methods described above for suggestively labeling a peptide with a rare isotope can be applied (in vivo labeling, enzyme Labeling, chemical labeling, etc.). One example of in vivo labeling is the incorporation of commercially available deuterated methionine CH ₃ —SCD ₂ —CD ₂ —CH— (NH ₂ ) —COOH, adding 4 amu to the total peptide mass. Alternatively, synthetic reference peptides could also be similarly contain deuterated arginine _{H 2 NC- (NH) -NH- (} CD 2) 3 -CD- (NH 2) -COOH), thus, the total 7 amu is added to the peptide mass. It should be clear to the person skilled in the art that all amino acids whose deuterated or ¹⁵ N or ¹³ C embodiment is present can be considered in this protocol. Another example of this approach is to proteolyze samples containing proteins with trypsin in the presence of H ₂ ¹⁸ O, but many other methods can be used. Thereby, in a preferred embodiment, the quantitative analysis of at least one protein in one sample comprises the steps of: a) preparing a protein peptide mixture in which the peptide carries a rare isotope (eg heavy isotope); b ) Adding a synthetic reference peptide carrying a natural isotope (for example, a light isotope) to this protein peptide mixture; c) separating the protein peptide mixture that also contains the synthetic reference peptide into fractions by primary chromatographic separation D) chemically and / or enzymatically modifying at least this reference peptide and its synthetic reference peptide counterpart; e) a reference peptide flagged by secondary chromatographic separation and a flagged synthesis reference. Isolating the peptide; f) a synthetic reference peptide The ratio of the peak height of the reference peptide in a sample comprising the steps and g) proteins determined by mass spectrometry which comprises the step of calculating the amount of protein is represented by the reference peptide.

  In another preferred embodiment, the reference peptide is also an identifying peptide at the same time. The above method can be equally well applied to this approach, but in step d) the reference peptide and its synthetic reference peptide will remain unmodified, and in step e) (reference peptide and The identified peptide (including its synthetic reference peptide) is isolated.

In another preferred embodiment, the quantitative determination of at least one protein in one single sample comprises digesting the protein mixture in said H ₂ ¹⁸ O into peptides with trypsin; b) natural isotopes Adding at least one synthetic reference peptide bearing a known amount to the resulting protein peptide mixture; c) fractionating the protein peptide mixture in a primary chromatographic separation; d) one or more specificities Chemically and / or enzymatically modifying each fraction on a specific amino acid (both peptides from a protein-peptide mixture and a synthetic reference peptide containing specific amino acids will be modified); e) second Isolate flagged peptides by chromatographic separation of Steps (these flagged peptides include both biological reference peptides and their synthetic reference peptide counterparts); f) the flagged peptides are analyzed by mass spectrometry to correspond to the reference peptides and their synthetic reference peptides Determining the relative amount of the object. Again, a similar approach can be followed with a reference peptide that is also an identified peptide.

  Similarly, the method described above can be applied in one mode in which a reference peptide is labeled with NIP and its synthetic reference peptide counterpart is labeled with a rare isotope.

  The above-described methods of the present invention for quantifying the amount of protein in a sample containing protein can be used to quantify from 1 to up to several hundred proteins in the sample. In order for all proteins to be quantified, at least one, preferably two or more reference peptides are required. In one particular embodiment, each synthetic reference peptide is added in an amount equimolar to the expected amount of its reference peptide counterpart.

  The methods provided in the present invention for quantifying at least one protein in a sample containing proteins can be widely applied to quantitate proteins with different advantages. For example, the present invention can be used to develop diagnostic assays that determine the level of one or more proteins in a sample.

  In another example, a reference peptide can be used to quantify specific known splice variants of a particular protein in a sample. If a specific splice variant is known from a specific protein and the purpose is to detect this splice variant, then a synthetic reference peptide corresponding only to this splice variant of the specific protein is synthesized. be able to. In practice, new junctions are often formed due to exon skipping, thereby allowing selection of specific reference peptides that do not occur in the parent protein but only occur in the splice variant. . However, in many cases, it is recommended to select more than one reference peptide to distinguish the splice variant in question from the parent protein. Similarly, it is common for a particular splice variant to be expressed in conjunction with the parent protein in the same cell or tissue, so that both are present in the sample. Often the expression levels of a particular splice variant and the parent protein are different. Using the detection and abundance between the reference peptides, the expression level between the splice variant and its parent protein can be calculated. In yet another example, it is well known that drugs can greatly affect the expression of specific proteins within a single cell. With current methods, it is possible to accurately measure the amount of a protein or protein of interest under different experimental conditions. Therefore, equivalent techniques such as genome utilization can be applied at the protein level including pharmacoproteomics and toxicoproteomics. While genetic markers of disease have received significant attention along with human genome sequencing, protein markers are more useful in many situations. For example, diagnostic assays based on reference peptides representing protein disease markers can be developed for essentially any disease in question. Most suitably, such disease markers are quantified in a cell, tissue or organ sample or body fluid including, for example, blood cells, plasma, serum, urine, semen, saliva, nipple aspirate fluid, synovial fluid or cerebrospinal fluid. Can do. At this time, according to the present invention, a reference peptide for a protein disease marker is used to determine whether the patient's disease is progressing rapidly or slowly, or whether the patient has a high probability of developing a certain disease. Can be used to monitor and even to monitor the efficacy of treatment. Indeed, in contrast to genetic markers such as SNPs, the level of protein disease markers representing specific diseases can change rapidly in response to disease modulation or progression. Reference peptides for protein disease markers, for example, according to the present invention, complex genetic diseases such as neuropsychiatric disorders including, for example, cancer, obesity, diabetes, asthma and inflammation, depression, depression, panic disorder and schizophrenia It can also be used to improve the diagnosis. Many of these diseases arise due to complex events reflected in numerous cellular and biochemical pathways and events. Thus, a number of protein markers may prove to correlate with these diseases. The present invention allows one to several hundred protein markers to be tracked simultaneously. The possibility of protein marker identification and relative and absolute quantification using the present invention could lead to a more accurate diagnostic subclassification.

  In another embodiment, the present invention is directed to a peptide sorter apparatus capable of performing the method of the present invention. As described herein, a method for analyzing a protein peptide mixture or a complex peptide mixture includes a primary chromatography step using a complete protein peptide mixture that separates the mixture into fractions and at least one of the peptides present in the fractions. Two consecutive chromatographic steps may be included, a second chromatographic step performed after chemical and / or enzymatic modification of specific amino acids. As described herein, the term “peptide sorter” is used to efficiently separate a flagged peptide from an unmodified peptide according to the present invention or alternatively identified from a modified peptide according to the present invention. An apparatus that efficiently separates peptides. In a preferred embodiment, during the second run, (i) the unmodified peptide stays at its original elution time and the flagged peptide is induced to undergo a shift in elution time or (ii) in reverse mode The same or very similar chromatographic conditions in the two chromatographic steps, such that the identified peptide stays in its original elution time and the modified peptide bulk is induced to undergo a shift in elution time. used. As described herein, the peptide sorter is particularly suitable for a pool of fractions obtained after run 1 and a second chromatographic step (e.g. a step where flagged peptides are separated from unmodified peptides or alternatively Means the optimal organization of the flagged peptide (or identified peptide) from each of the fractions of run 1 to be separated from the identified or modified peptide to accelerate the isolation.

  One approach for isolating and identifying flagged peptides isolated from protein-peptide mixtures is to collect all fractions independently from the primary chromatographic separation, and chemically and / or within each of the fractions. Alternatively, an enzymatic modification is performed and all fractions are rerun independently, within the same chromatographic conditions and on the same or substantially similar column. In effect, each independently run secondary run flagged peptide is collected and passed to an analytical instrument such as a mass spectrometer. However, such an approach requires significant chromatographic time and occupies a lot of work time on a mass spectrometer. In order to obtain a more efficient and economical use of both chromatographic instruments and mass spectrometers, the present invention can be used between flagged peptides from different fractions and flagged peptides from one fraction. Provided is the use of a peptide sorter that allows pooling of multiple fractions of primary chromatographic separation while avoiding elution overlap between unmodified peptides from one or more other fractions, and in reverse mode, Multiple fractions of primary chromatographic separation while avoiding elution overlap between identified peptides from different fractions and between identified peptides from one fraction and modified peptides from one or more other fractions Peptide Sorter that enables pooling There is provided the use.

  The general principle of the system for sorting peptides can be illustrated as follows. In each fraction obtained from the primary chromatography step, the flagged peptide elutes in a completely different manner than the unmodified peptide. Isolated from the primary chromatographic run (w1) if alteration of the amino acid (s) in the flagged peptide induces a shift in the elution of the flagged peptide at the lower limit of δmin and the upper limit of δmax The elution window of each fraction made can be equal to δmin, but is preferably less than δmin / 2 to allow a completely different elution of the maximum number of flagged and unmodified peptides within a fraction. is there. The primary run is divided into fractions referred to here as window w1 between times t3 and t4. In a non-limiting example, w1 is 1 minute (FIG. 1A). An example of a chromatographic shift resulting from the conversion of a peptide into its modified derivative is depicted in FIG. 1B. Thereby, this concept is illustrated by arbitrarily selecting the 1 minute fraction eluting between t3 and t4. Peptides from this fraction modified show a hydrophilic shift (shift for each modified peptide) expressed as δp. Since the effect of the modification is always the same for all peptides derived from the selected fraction, δp shows a different value for all modified peptides, so between two external values: δmin and δmax Will fluctuate (thus δmin ≦ δp ≦ δmax). Let t1 and t2 be the time at which the sorted flagged peptides start and stop elution, and t3 and t4 be the time surrounding the selected fraction, δmin = t3-t2, δmax = t4-t1 Become. Thereby, the window (w2) in which the sorted peptides elute can be represented by the hydrophilicity shift and the selected fraction size (w1). w2 = t2−t1 or w2 = δmax−δmin−w1 (Equation 1).

If multiple fractions of the primary run are combined (pooled), during the second run in the pooled fraction, the peptides that have been flagged sorted from one selected fraction are: It is important not to co-elute with one unmodified peptide of the preceding fraction. This is schematically represented in FIG. Thereby t′1 should be measured from t4 and start at time difference w3. W3 should not be considered zero, as some spread is always observed for unmodified peptides during the secondary run. This means that the elution time of the next fraction, t'3, will be expressed as:
t′3 = t3 + w1 + w3 + w2 + δmin
Or t′3 = t3 + w1 + w3 + w2 + δmax−w1−w2
Or t′3 = t3 + w3 + δmax
Or t′3−t3 = Δt = w3 + δmax (Equation 2)

  The space between two consecutive fractions that can preferably be pooled thereby is determined by the spacing between the unmodified peptide of a given fraction and the flagged peptide of the next fraction and δmax. The Thereby, if δmax = 7 minutes and w3 = 5 minutes, the fractions of the primary run that can be preferentially combined for the secondary run are separated by 12 minutes (eg w1 is 1 minute) If equal, fractions 10, 22, 34, etc.). These values apply when δmax and δmin remain constant over the entire slope. Depending on the chromatographic conditions, the δp value can vary slightly across the fraction. For example, it was observed that the hydrophilicity shift for the modified methionine-peptide was slightly less for the more hydrophobic peptide compared to the more hydrophilic one eluting at an earlier time point. Within the TFA / acetonitrile system, this regression is limited and can therefore be easily corrected. However, for other chromatographic conditions, this regression is even more pronounced and is therefore taken into account. Accordingly, a correction factor λn (as shown in Example 18) is provided. λn is the correction factor for δp at a given fraction (η) where the concentration of solvent B is shown as ConcBn. If a linear correlation exists between λn and ConcBn, this will be expressed as λn = a, ConcBn + b [Equation 3]. Using the TFA / acetonitrile system described in Example 18, a C18RP-HPLC column and a hydrophilic film resulting from oxidation of the methionine peptide, it was determined that a = 0.002 and b = 1.002. Thus, in this example, for a fraction containing 10% solvent B, λ10 = −0.002.10 + 1.002 = 1, and for a fraction containing 50% solvent B, λ50 = −0.002. -50 + 1.002 = 0.902. This means that w2 is 7 minutes-2 minutes-1 minutes = 4 minutes when δmin = 2 minutes and δmax = 7 minutes in the fraction with 10% solvent B. This value for w2 in combined or pooled mode in fractions containing 50% solvent B is: w2c = 7 minutes.0.902-2 minutes.0.902-1 = 3. 51 minutes.

  Thereby, to adjust the sorting system, δmax and δmin are determined for the early and late elution fractions, respectively, and then set as δmax taken from the early fraction and δmin taken from the late FX. As a result, w2c: δmax. λ (early fraction) −δmin. λ (late fraction) -w1 [Equation 4].

In one example,
w2c = 7 minutes-1.8 minutes-1 minute = 4.2 minutes

  After using a constant w2c-value throughout the entire sorting process, regression of δmax and δmin can also be used to select a fraction of the primary run to achieve better sorting efficiency. This may be true if the shift is strongly affected during the solvent B gradient. Assuming the values a and b in Equation 3 are a = −0.02 and b = 1.2, δmin = 2 min, δmax = 7 min, the shift in fraction 10 (10% solvent B) is δmax = 7 minutes and w2 = 4 minutes. If δmax and δmin are not affected during the run, the next fraction should be at 22 minutes. However, assuming that the values for a and b in Equation 3 are a = −0.02 and b = 1.2, λ22 = −0.02 × 22 + 1.2 = 0.766, δmax22 = 7 × 0.76 = 5.32 minutes. Also, t′3 = 10 minutes + 5 minutes + 5.32 minutes = 20.32 minutes. Thereby, the next fraction can now be fraction 21 instead of fraction 22 and the new λ21 is now λ21 = 0.78, δmax × 21 = 7 × 0.78 = 5.46 = 5.46 minutes It becomes. Thereby, t′3 can be considered at least 10 minutes + 5 minutes + 5.46 minutes = 2.46 minutes. If the next selected fraction is fraction 21, it is possible to recalculate which next fraction will most closely follow the preceding one. This is fraction 31, for which λ31 is 0.58 and δmax = 4.06 minutes. This results in t ″ 3 = 21 minutes + 5 minutes + 4.06 minutes = 30.1 minutes. According to the same calculation, it includes fraction 39 where λ39 = 0.42 and δmax = 2.94 minutes. So t ″ ′ 3 = 31 minutes + 5 minutes + 2.94 minutes = 38.94 minutes and so on. To illustrate the principle of the peptide sorter, an example was performed on a flagged peptide, with a simple fraction with an elution window w1 equal to x / 2 and no regression (assuming a constant shift throughout the gradient). Be released. If the total run 1 elution window of all peptides from the protein peptide mixture is equal to 20x, then 40 fractions with a window of x / 2 (first fraction: 0 to x / 2; second fraction : X / 2 to x; third fraction: x to 3x / 2, ...) is recovered. In the simplest approach, all fractions are individually subjected to chemical and / or enzymatic amino acid modification steps and the peptide is subjected to run 2 under chromatographic conditions substantially similar to run 1. Run 2 separates the flagged peptide from the unmodified peptide. To limit chromatography and analysis time, the procedure has been optimized by pooling the fractions obtained from Run 1. Pooling can be performed in the primary fraction prior to the modification reaction, otherwise it can be performed in the modified fraction. A modified fraction is a fraction in which the peptide has undergone chemical and / or enzymatic modification in accordance with the present invention.

  In the example, the pooling is done in the fraction before the modification reaction. After the primary chromatography run, the following fractions are pooled: fraction 1 (0 × x / 2), and fractions 8 (7x / 2-4λ), 15 (7x-15x / 2), 22 (21x / 2) ~ 11x), 29 (14x-29x / 2), and 36 (35x / 2-18x). In a similar manner, fraction 2 is pooled with fractions 9, 16, 23, 30 and 37; fraction 3 is pooled with fractions 10, 17, 24, 31 and 38; fraction 4 is fractions 11, 18, 25, 32 Fraction 5 is pooled with fractions 12, 19, 26, 33 and 40; Fraction 6 is pooled with fractions 13, 20, 27 and 34; Fraction 7 is fractions 14, 21, 28 and 35 Pooled with. Seven pools are chemically and / or enzymatically modified on at least one specific amino acid, and each of the seven pools is separately separated into run 2 under chromatographic conditions substantially similar to primary chromatographic separations. Attached. By selecting the correct combination of fractions within each pool, the flagged peptides elute within a window that is completely different from the time known for the unmodified peptide to elute, and within the same pool. There is no overlap between the flagged peptides from the different fractions. A specific amino acid mutation in the flagged peptide induces a forward shift on the hydrophobic separation column, and the value of this shift varies between x and 2x (this is the value for δmin = x / 2) , Δmax = 5x / 2, implying w1 = x / 2 and w3 = 7x / 2), in a non-limiting example, the flagged peptide in the first pool is a fraction, for example [-2x to -x / 2], [3x / 2 to 3x], [5x to 13x / 2], [17x / 2 to 10x], [12x to 27x / 2] and [31x2 to 17x] Will be collected. A similar approach is followed for pools 2-7. Thus, in this example, instead of 40 reruns, only 7 secondary runs need to be performed on the pool. Peptides that are flagged to elute during this secondary run can be transferred directly into, for example, an ion source of an on-line connected mass spectrometer for immediate identification. The pool strategy described above is a non-limiting example. It will be apparent to those skilled in the art that similar strategies can be developed to create more or fewer pools and similar strategies can be applied to the identified peptides. Selection of the number of pools, among other things, optimizes (i) interval shifts induced by chemical or enzymatic modification, ii) elution windows of fractions recovered from primary chromatographic separations, and iii) chromatographic and analytical times Depends on the need to convert. The present invention also provides for the use of a parallel column sorter. In a parallel column sorter, a single column based method is performed using a fixed number of columns operating in parallel (ie synchronously). The parallel sorter contains a certain number of identical columns that are run under exactly the same conditions (flow rate, gradient, etc.).

  The general principle of the parallel sorter is to generate 12 peptide fraction pools, where δp is between x / 2 and 5x / 2, the hydrophilic shift between the flagged and unmodified peptides, This can be explained by a non-limiting example. If the total elution window of all peptides from the primary chromatography run is equal to 20x, 40 fractions with x / 2 windows are collected. Thereby, after the primary chromatographic run, the following fractions can be pooled: namely fractions 1 (0-x / 2), and fractions 13 (6x-13x / 2), 25 (12x-25x / 2) ) And 37 (18x-37x / 2). Similarly, fraction 2 is pooled with fractions 14, 26 and 38; fraction 3 is pooled with fractions 15, 27 and 39; fraction 4 is pooled with fractions 16, 28 and 40; fraction 5 is fractions 17 and 29 Fraction 6 is pooled with fractions 18 and 30; fraction 7 is pooled with fractions 19 and 31; fraction 8 is pooled with fractions 20 and 32; fraction 9 is pooled with fractions 21 and 33; Fraction 10 is pooled with fractions 22 and 34; fraction 11 is pooled with fractions 23 and 35; fraction 12 is shared with fractions 24 and 36. It is pooled. The 12 pools are then chemically and / or enzymatically modified on at least one selected amino acid. In an alternative approach, each fraction is first subjected to modification, and pooling is performed on the modified fraction. Table II contains calculations of theoretical shifts for 12 flagged peptide pools. Each of the twelve modified pools (ie, the pool containing the modified fraction) is subjected to run 2 under chromatographic conditions that are equal to or at least very similar to the primary chromatographic separation on a single column sorter. In each case, there is a 9x / 2 "empty" elution window between the fractions containing the flagged peptide (present in one pool). This 9x / 2 empty elution window does not elute any flagged peptides within this window, and no flagged peptides can be sent to the appropriate analyzer, so chromatographic separation and analysis It is a “dead interval” for the device. A parallel column sorter most suitably performs a secondary chromatographic run with substantially similar conditions (flow rate, gradient etc.) on 2, 3, 4 or more columns at the outlet of the parallel sorter. Is a device directly connected to the analyzer. A parallel column sorter divides the chromatographic separation time normally required for a series of serial single columns by approximately the number of columns used in the parallel sorter. In a non-limiting example where the parallel column sorter consists of three columns, the modified pool is rerun in parallel with the preferred combination of modified pools. Thereby, the advantage of using a parallel column sorter is not only that the overall peptide sorting time can be significantly reduced, but also has been modified in such a way that the detection of flagged peptides can be carried out continuously. It may also mean that the number of dead intervals between the selection of peptides flagged from fractions is limited. As illustrated in Table II, for the above non-limiting examples, the preferred combination of modified pools is that the modified pools 1, 5 and 9 are three in the first run. The modified pools 2, 6 and 10 are loaded onto three parallel columns in the second run, and the modified pools 3, 7 and 11 are placed in the third run. In this case, the modified pools 4, 8 and 12 are put on the three parallel columns in the fourth run. In the above combination of modified pools, there is an almost perfect alignment between the intervals at which the flagged peptides elute from the parallel column in each of the four runs. If columns I, II and III are started simultaneously, the flagged peptides from column I, pool I, fraction 1 will elute first in windows -2x to -x / 2. The next flagged peptide stream comes from column II, pool 5, fraction 5; these flagged peptides will elute in windows 0-3x / 2. These are followed by the flagged peptides from column III, pool 9 and fraction 9 eluting at www 2x to 7x / 2. Subsequent flagged peptides elute from column I, pool 1, fraction 13 at www4x to 11x / 2, etc. (to avoid the possibility of overlap between flagged peptides from different pools, x / 2 window was introduced between each two flagged peptide elution windows). In the peptide sorter, the flagged peptides eluting from columns I, II and III are continuously transferred to an analyzer such as a mass spectrometer. The fact that the flagged peptides in each run elute without interruption leads to a continuous flow of peptides into the analyzer. Once the first run is complete, the second run can be started, followed by the third and fourth runs. The pool strategy described above is a non-limiting example. For those skilled in the art, other combinations of the number of pools and parallel columns allow similar results, i.e., continuous chromatography of peptides flagged immediately coupled to continuous analysis of peptides in a mass spectrometer, for example. It will be clear that elution can be guided. The choice of the number of pools and columns is, among other things, i) the interval δp induced by chemical or enzymatic modification, ii) the elution ww and iii) chromatographic and analytical times of the fraction recovered from the primary chromatographic separation It depends on the need to optimize. Similarly, it will be apparent to those skilled in the art that this parallel column approach can be applied to the isolation of identified peptides.

  In another aspect of the invention, a multi-column peptide sorter is provided. Such a multi-column peptide sorter has been created and basically consists of a fixed number of parallel column sorters operating in a combined parallel serial mode. Such a parallel sorter basically includes a set of z columns y times, where the z columns are connected in parallel. In a non-limiting example, a multi-force ram sorter with y = 3 and z = 3 is a 9 column sorter. The three parallel column sets are called A, B, and C. The individual columns of A are called I, II and III, the individual columns of B are called I ', II' and III '; the individual columns of C are I ", II" and III " One set of parallel columns operates with a constant delay (referred to as θ) relative to the preceding set, so parallel sorter B has a delay of θ in relation to parallel sorter A. The parallel sorter C starts with a delay of θ after the start of the parallel sorter B and with a delay of 2θ after the start of the parallel sorter A. In a multi-column sorter, one run of modified peptides is started. It is important to point out that only one fraction is processed at a given time per column, so that in the 9 column sorter example the flagged peptide (or identification) Nine fractions of peptide) are processed at the same time, which is different from the two previous sorters (ie 1 column peptide sorter and parallel sorter) where multiple modified fractions are strategically pooled and loaded simultaneously. Since only one fraction of peptides (or identified peptides) flagged at that time on a multi-column sorter is processed, control of flow accuracy (ie secondary chromatography step) is as important as the sorter described above. Another advantage of the multi-column sorter is that it separates flagged peptides from unmodified peptides when the chromatographic shift of flagged peptides varies significantly across different fractions. It is in that it is well adapted. Similarly, multiple column sorters are well adapted for separating identified peptides from modified peptides.

  The mechanism of the multiple column sorter is illustrated as a non-limiting example for a 9 column peptide sorter that uses a modified fraction elution window as shown in Table II. The modified fraction 1 is charged on the column I, the modified fraction 13 is charged on the column II, and the modified fraction 25 is charged on the column III. System B (columns I ′, II ′ and III ′) is charged with modified fractions 2, 14 and 26, respectively, and system C (columns I ″, II ″ and III ″) is respectively modified fraction 3 .15 and 27. As can be seen from Table II, the three columns of System A are started simultaneously, and all three columns of System B start with a delay θ = 3x / 2 However, if all three columns in System C start with a delay of 2θ = 3x (relative to System A), the dead time during elution of the flagged peptide is minimal. For example, if the sorter is run according to the setpoint values described above, the flagged peptides from System A, Column I, and Fraction 1 will first elute at a predetermined ww-2x to -x / 2. Subsequently, the flagged peptide from system B, column I ′, fraction 2 elutes at www0-3x / 2, followed by system C, column 1 ″, fraction 3 Flagged peptides elute at www2x-7x / 2, then flagged peptides from System A, column II, fraction 13 elute at ww4x-11x / 2, and so on. Further, the complete sorting of the fractions presented in Table II is as follows: Run 1: System A (1, 13, 25), System B (2, 14, 26), System C (3, 15, 27); 2: System A (4, 16, 28), System B (5, 17, 29), System C (6, 18, 30); Run 3: System A (7, 19, 31), System B (8, 20, 32), System C (9, 21, 33); Run 4: System A (10, 22, 34), System B (11, 23, 35), System C (12, 24, 36) and Run 5 : System A (37), System B (38), System C (39, 40) can be implemented in five runs. It will be apparent to those skilled in the art that other combinations of parallel and serial columns can lead to similar results and that a multi-column peptide sorter is equally applicable to the isolation of identified peptides. . The number of columns, their arrangement and the selection of the fractions loaded on the column are, among others, i) the interval δp induced by chemical or enzymatic modification, ii) the elution window of the fractions recovered from the primary chromatographic separation and iii) depends on the need to optimize chromatographic and analysis times.

  It will be apparent to those skilled in the art that a peptide sorter implementing the method of the present invention can function fully automatically using commercially available autoinjectors, HPL-instruments and automatic fraction collectors. Therefore, such examples of peptide sorters should not be considered exhaustive. Multiple variations including electrophoresis and ion exchange chromatography systems are equally feasible. For completeness, a peptide sorter for sorting identified peptides can be designed based on the same principle.

  The exemplary embodiments further provide a system for implementing the above-described proteome analysis method in a selective and efficient manner. As discussed, the primary chromatography column performs an initial separation of the complex peptide mixture. The primary chromatography column separates the complex peptide mixture into at least two fractions under a defined set of conditions. For example, a primary chromatography column separates protein peptide mixtures by eluting the column with a predetermined solvent gradient and a predetermined flow rate. The fractions resulting from the primary chromatographic separation can be strategically pooled to mix fractions with completely different elution times into a plurality of pooled fractions as described above. The pooled fractions can then be modified to result in a set of modified peptides and a set of unmodified peptides for each fraction. According to one variant embodiment, the fractions are first modified using the method described above and then strategically pooled into a set of pooled fractions, where each of the pooled fractions is The fraction comprises a set of modified peptides and a set of unmodified peptides. In secondary chromatographic separation, the modified peptide is separated from the unmodified peptide. At this time, the isolated peptide can be analyzed to identify the protein.

  Secondary chromatographic separations can be performed using a single column peptide sorter 10, as illustrated in FIG. According to an exemplary embodiment, a single column peptide sorter 10 operates in turn with a primary chromatography column and includes a secondary chromatography column 11. According to an exemplary embodiment, the secondary chromatography column 11 is substantially the same in type, size, shape and other parameters as the primary chromatography column. The exemplary secondary chromatography column 11 further functions under substantially similar chromatography conditions. For example, according to an exemplary embodiment, the secondary chromatography column is the same or substantially similar to the solvent gradient and the same or substantially the solvent gradient used to effect the separation within the primary chromatography column. Elute at similar flow rates. The exemplary peptide sorter 10 further includes a solvent system that includes a solvent pump 12 coupled to at least one solvent tank. The solvent pump 12 provides a predetermined solvent gradient for the secondary column 10. A sample injector 13 is provided for introducing fractions or pooled fractions into the column for separation. The system 10 for sorting peptides further includes a set of inlet valves 14 for controlling and directing solvent and sample flow to the secondary column inlet 15. The system 10 for sorting the peptides of FIG. 9 further controls the flow from the outlet 6 of the secondary column 10 and allows the ion source of the waste container 17, the fraction collector 18 and / or the analyzer 19 connected online. An outlet valve set 51 for guiding the effluent from the secondary column 10 between them is included. A valve control system 50 is provided to control and guide the operation of the valves 14, 51. According to an exemplary embodiment, the analyzer 19 includes a mass spectrometer, although one skilled in the art can utilize any suitable analyzer for identifying proteins such as those described herein. You will recognize that. As discussed, the modified peptide elutes in a completely different manner from the unmodified peptide in the secondary column 10 so that the modified peptide can be isolated and identified. For those skilled in the art, it is clear that the same principle (column) can be used to separate the identified peptide from the modified peptide.

  Although the column utilized to provide the secondary chromatography step is illustrated as being completely different from the primary column, those skilled in the art will perform the primary and secondary chromatography steps of the exemplary embodiment. You will recognize that you can use a single column to do that. For example, the complex mixture can be separated into fractions using a given chromatography column. A given column can be cleaned and then reused for secondary chromatography steps.

  According to a variant embodiment, the separation of the modified fractions can be carried out using a system for sorting parallel column peptides, as illustrated in FIGS. 12a and 12b. As discussed, complex mixtures are first separated using a primary chromatography column, modified using the methods described above, and strategically pooled before or after the modification step. The modified pooled fraction is then subjected to a second chromatographic step resulting in separation of the modified and unmodified peptides. The system 20 for sorting parallel column peptides illustrated in FIG. 12a significantly improves the efficiency and speed of the secondary chromatography step. As illustrated, the parallel column system 20 includes a plurality of substantially identical secondary chromatography columns 21a, 21b, 21c connected in parallel. The secondary chromatography columns 21a, 21b, 21c of the parallel column system 20 are the same or substantially the same as the primary chromatography column used to perform the primary separation of the complex mixture in terms of size, shape and other chromatographic parameters. It is similar. According to an exemplary embodiment, the system for sorting peptides includes three secondary columns. However, those skilled in the art will recognize that any suitable number of chromatography columns connected in parallel can be utilized and that the present invention is not limited to the exemplary embodiment of three columns. That would be true. The same mechanical principle can be applied to isolate the identified peptide.

  As shown in FIG. 12a, the parallel column system 20 includes a set of solvent systems 22a corresponding to each column 21a, 21b, 21c, respectively. Each solvent pump is connected to a set of solvent tanks and provides a predetermined solvent gradient for the corresponding secondary column. The system 20 for sorting parallel column peptides further includes a sample injector 23 coupled to the inlets 25a, 25b, 25c of the secondary columns 22a, 22b, 22c to introduce the sample into one or more secondary columns. It is out. A set of inlet valves 24 are provided to control the solvent and sample streams and direct them to the selected secondary column. Also, the elution from the inlets 26a, 26b, 26c of the secondary columns 20a, 20b, 20c between the ion sources of the analyzer 29 connected online such as the waste container 27, fraction collector 28 and / or mass spectrometer A set of outlet valves 53 are provided to control and direct the objects. In order to control the operation of the valves 24 and 53, a valve control system 55 is provided.

  According to a variant embodiment of the system for sorting parallel column peptides shown in FIG. 12b, the solvent gradient for the secondary parallel columns 20a ', 20b', 20c 'in the system 20' for sorting peptides is shown. A single solvent pump 58 is utilized to provide. The system for sorting the peptides of FIG. 12b is substantially similar to the system for sorting the peptides of FIG. 12a, except for the solvent system. As illustrated, a single solvent pump 58 is utilized to pump the solvent mixture from the solvent tank to the parallel secondary column. In the embodiment illustrated in FIG. 12b, a controlled splitter system including a set of flow regulators 52 directs solvent flow from the solvent pump 58 to the parallel secondary columns 20a ', 20b', 20c '. Used for controlling and guiding.

According to yet another embodiment shown in FIG. 13, the system 30 for sorting alternative peptides for performing the secondary chromatography step of the present invention operates in a serial / parallel combination mode. Contains multiple parallel column sets. According to an exemplary embodiment, the system for sorting the peptides of FIG. 13 includes a plurality of serially connected secondary column sets 41, 42, 43. Each secondary column set 41, 42, 43 includes a plurality of secondary columns 41a, 41b, 41c, 42a, 42b, 42c, 43a, 43b, 43c connected in parallel.
A person skilled in the art is not limited to the illustrated number of columns and column sets for sorting exemplary peptides and is optional for performing the secondary chromatography steps of the exemplary embodiments. It will be appreciated that any suitable number of parallel columns and serial column sets can be utilized.

  In the system 30 for sorting the peptides of FIG. 13, a single modified fraction of peptides produced from a primary chromatography run is processed at a given time per column. The fractions are loaded one at a time on the selected column, and a solvent gradient is provided for each respective column to effect separation of the modified peptides in each fraction from unmodified peptides. Sample injectors 13a, 13b, and 13c are provided and connected to each of the secondary column sets 41, 42, and 43, respectively, for introducing peptide fractions into selected secondary columns within the secondary column set. In order to provide a predetermined solvent gradient at a predetermined time for each column set, a solvent system including solvent pumps 12a, 12b, 12c is provided in each secondary column set 41, 42, 43, respectively. Provided and connected. A set of inlet valves 44 controls and directs the solvent flow from the solvent pump and the sample flow from the sample injector to the secondary column inlets in the secondary column sets 41, 42, 43. Secondary column outlets 41a, 41b, 41c, 42a are connected to the waste container 47, the fraction collector 48 and / or the ion source of the analyzer 49 connected online to guide the effluent from the secondary column. . A valve control system 50 is provided for controlling and guiding the operation of the valves 14, 51.

  The solvent pumps 12a, 12b, 12c are configured to initiate a predetermined solvent gradient for each column set 41, 42, 43 at a selected time period. For example, the first solvent pump 12a is the first in the column set 41 at a first predetermined time to effect separation of each fraction in the secondary columns 41a, 41b, 41c of the first set 41. Initialize the appropriate solvent gradient. A solvent gradient is developed across each secondary column 41a, 41b, 41c in the first set. After the selected delay, the second solvent pump 12b initializes the same or substantially the same solvent gradient in the second set of secondary columns 42 at a second predetermined time. . The second solvent system develops a solvent gradient across each secondary column 42a, 42b, 42c to provide separation of each fraction within the secondary columns 42a, 42b, 42c in the second set 42. Finally, after a selected delay, the third solvent pump 12C initially initiates the same or substantially the same solvent gradient in the third secondary column set 43 to provide separation of the third fraction set. Turn into. The described arrangement allows the fraction collector 48 and / or on-line analysis of a continuous stream of separated and isolated peptides to identify the modified peptide in the fraction and the protein corresponding to the modified peptide. Provided to the ion source of the device 49.

  The system for sorting the peptides of FIG. 13 can be implemented using secondary column sets 41a, 41b, 41c, 42a, 42b, 42c, 43a, 43b, 43c that are substantially smaller than the primary column. Similarly, the secondary columns 41a, 41b, 41c, 42a, 42b, 42c, 43a, 43b, 43c may be formed of a less expensive disposable material. In this way, the system 30 for sorting the peptides of the illustrated embodiment not only significantly improves the speed and efficiency of the proteome analysis of the present invention, but also reduces the cost of performing the analysis. The mechanical principles described above can be used to separate flagged peptides from unmodified peptides or to separate identified peptides from modified peptides.

  Thereby, in another embodiment, the present invention aims at separating a protein peptide mixture into a plurality of fractions under a defined set of conditions in a system for sorting peptides, Each fraction is then subjected to at least one amino acid modification to produce a flagged peptide, and the modified fractions are pooled into a set of pooled fractions, each pooled A primary chromatography column wherein the fraction comprises at least two modified fractions; and b) a first secondary chromatography column for separating the first pooled fraction and a second pooled First secondary chroma to separate the fractions Isolate flagged peptides under conditions that include at least one second secondary chromatography column placed in parallel with the tomography column and are substantially identical to a defined set of conditions So that there is no elution overlap between (i) flagged peptides from different fractions within a pool or between pools and (ii) flagged and unmodified peptides A system comprising a set of secondary chromatography columns.

  In yet another embodiment, the object is to separate the protein peptide mixture into a plurality of fractions under a defined set of conditions, whereby each fraction has been subsequently modified with at least one amino acid modification Peptides and unmodified peptides will be generated, and the modified fractions are pooled into a set of pooled fractions, each pooled fraction containing at least two modified fractions In parallel with a first chromatography column; and a first secondary chromatography column for separating a first pooled fraction and a first secondary chromatography column for separating a second pooled fraction. At least one second secondary cluster arranged Isolation of identified peptides under conditions that include a matography column and are substantially identical to a defined set of conditions, thereby (i) between or between identified peptides from different fractions within one pool And (ii) providing a system for sorting peptides, including a set of secondary chromatography columns ensuring that no elution overlap exists between the identified peptide and the modified peptide .

  In another embodiment, the system further includes an outlet to a second chromatography column set for collecting eluate from the first secondary chromatography column and the second secondary chromatography column.

  In another embodiment, the system further includes an analyzer coupled to the outlet.

  In another embodiment, the system further includes a waste container coupled to the outlet for collecting waste from the secondary chromatography column set.

  In yet another embodiment, the system is further coupled to a secondary chromatography column set for injecting a pooled fraction into one of the first secondary column and the second secondary column. Sampled injector.

  In yet another embodiment, the system further includes a sample injection valve set for directing a pooled fraction from the sample injector to one of the first secondary column and the second secondary column.

  In yet another embodiment, the system further includes a solvent system for providing a solvent gradient for the secondary chromatography column set.

  In yet another embodiment, the solvent system includes a first solvent pump for providing a solvent gradient for the first secondary chromatography column and a second secondary chromatography column. A second solvent pump for providing a solvent gradient is included.

  In another embodiment, the solvent system includes a solvent pump coupled to the first secondary chromatography column and the second secondary chromatography column, solvent flow to the first secondary chromatography column. A first flow controller for adjusting the flow rate and a second flow control device for adjusting the solvent flow to the second secondary chromatography column.

  In another embodiment, the system further includes a fraction collector for collecting the eluate from the secondary chromatography column set.

  In yet another embodiment, the system further includes a valve control system for controlling the sample injection valve set.

  In yet another embodiment, within the system, the first and second secondary chromatography columns are substantially identical to the primary column.

  In yet another embodiment, within the system, a first solvent gradient is applied to the primary column to effect separation of the protein peptide mixture and the first solvent to effect separation of the pooled fractions. A second solvent gradient that is substantially the same as the gradient is applied to the secondary column.

  In another embodiment, the present invention provides a first chromatography column set comprising a first chromatography column and a second chromatography column disposed substantially in parallel therewith, a first chromatography column. A first sample injector for providing a sample to the set; a first solvent system for providing a predetermined solvent gradient to the first chromatography column at a first predetermined time point; A second chromatography column set comprising a chromatography column and a fourth chromatography column arranged substantially in parallel therewith, a second sample injector for providing a sample to the second chromatography column set, And a second chromatogram at a second predetermined time point Provides a system for sorting peptides comprising a second solvent system for providing a solvent gradient to a predetermined relative fee column.

  In yet another embodiment, the sorting system further includes a waste container coupled to the output ends of the first and second chromatography column sets for recovering the waste from the chromatography column. In yet another embodiment, the sorting system further includes a fraction coupled to the output ends of the first and second chromatography column sets for collecting eluate from the column at predetermined intervals. Includes collectors. In yet another embodiment, the sorting system further includes an analyzer coupled to the output ends of the first and second chromatography columns. In another embodiment, the sorting system further includes an inlet valve set coupled to the inlet of the chromatography column to control the inlet of the chromatography column. In yet another embodiment, the sorting system further includes an outlet valve set coupled to the outlet of the chromatography column to direct the eluate from the column to one of the waste container, fraction collector and analyzer. Including. In yet another embodiment, the sorting system further includes a valve control system for controlling the inlet valve set and the outlet valve set.

  In another embodiment, the present invention provides a fraction set of protein peptide mixtures; for sorting peptides comprising a first parallel chromatography column set and a second parallel chromatography column set Providing a system, applying a first fraction set of a protein peptide mixture to a first column set; applying a second fraction set of a protein peptide mixture to a second column set; Providing a solvent gradient within the first column set at a first predetermined time to initialize the separation; and a first predetermined to initialize the separation of the second fraction set. Dissolved in the second column set at a second predetermined time following the time. Comprising providing a gradient, to provide the peptide separation methods. In yet another embodiment, the invention further includes directing the eluate from the first column set to one of the waste container, fraction collector, and analyzer. In yet another embodiment, the invention further includes directing the eluate from the second column set to one of the waste container, fraction collector, and analyzer.

  In another embodiment, the present invention provides a method for isolating flagged peptides from a protein peptide mixture, comprising: (a) providing a primary chromatography column for separating the protein peptide mixture; ) Injecting the protein peptide mixture into the primary chromatography column to separate the protein peptide mixture into a set of fractions under a defined set of conditions; (c) modifying at least one of the fractions in the fraction set; Forming a set of modified fractions, wherein the modified fraction comprises a flagged subset of peptides and a subset of unmodified peptides; (d) a first modified fraction; Pool the second modified fraction Forming a first pooled fraction, wherein i) between the flagged peptides from the first and second modified fractions, and ii) the unmodified peptides of said fractions (E) pooling the third modified fraction and the fourth modified fraction to form a second pooled fraction; no elution overlap between the flagged peptides; Wherein i) an elution overlap between the flagged peptides from the third and fourth modified fractions, and ii) between the unmodified peptide and the flagged peptides of the fractions. (F) a first secondary chromatogram to separate a flagged subset of peptides from a subset of unmodified peptides; Providing a fee column; and (g) subjecting the first modified fraction to secondary chromatography under a defined set of conditions to isolate a subset of flagged peptides within the second modified fraction. A method is provided that includes separating a first pooled fraction using a column.

  In another embodiment, the present invention provides a method for isolating flagged peptides from a protein peptide mixture, comprising: (a) providing a primary chromatography column for separating the protein peptide mixture; ) Injecting the protein peptide mixture into the primary chromatography column to separate the protein peptide mixture into a set of fractions under a defined set of conditions; (c) modifying at least one of the fractions in the fraction set Forming a set of modified fractions, wherein the modified fraction comprises a modified subset of peptides and a subset of identified peptides; (d) a first modified fraction and a first Pool 2 modified fractions Forming one pooled fraction, wherein i) between the modified peptides from the first and second modified fractions and ii) the identified peptide of the fraction and the modified peptide (E) pooling the third modified fraction and the fourth modified fraction to form a second pooled fraction, wherein no elution overlap exists between: I) between the identified peptides from the third and fourth modified fractions, and ii) no elution overlap between the identified peptides of the fractions and the modified peptides; (f) the identified peptides Providing a first secondary chromatography column to separate a modified subset of peptides from the subset of; and ( ) Separating the first pooled fraction using a secondary chromatography column under a defined set of conditions to isolate the first modified fraction from a subset of identified peptides in the second modified fraction A method comprising the steps of:

  In yet another embodiment, the present invention further provides defined conditions for isolating flagged peptides or subsets of identified peptides within the third modified fraction and the fourth modified fraction. Separating a second pooled fraction using a first secondary chromatography column under set.

  In yet another embodiment, the method further comprises: (h) substantially separating the modified peptide subset from the unmodified peptide subset in one fraction with a first secondary chromatography column. Providing a second secondary chromatography column arranged in parallel; and (i) isolating a third modified fraction and a subset of modified peptides in the second modified fraction. Preferably, separating the second pooled fraction using a second secondary chromatography column under a defined set of conditions.

  In yet another embodiment, the method further comprises directing the flagged or identified peptide to an analyzer.

  In yet another embodiment, the method further comprises identifying the identified peptide or flagged peptide and its corresponding protein using an analytical device in combination with a database search.

  In the following, a description is provided that provides more information about some of the different steps of the present invention.

I. Preparation of protein peptide mixtures Protein peptide mixtures derived from samples containing proteins (protein peptide mixtures) are obtained by methods described in the art such as chemical or enzymatic resolution or digestion. In a preferred embodiment, the protein is digested by proteolytic enzymes. Trypsin is a particularly preferred enzyme because it splits at the lysine and arginine sites to produce a charged peptide that is typically about 5-50 amino acids long and has a molecular weight of about 500-5000 daltons. Such peptides are particularly suitable for analysis by mass spectrometry. Similarly, a non-restrictive list of proteases that can be used in the present invention includes Lysobacter enzymogenes endoproteinase Lys-C, Staphylocolococus aureus endoproteinase Glu-C (V8 protease), Pseudomonos fragi endoproteinase Asp-N, and Clostri. There is a pine. In the present invention, proteinases having lower specificities such as Bacillus subtilis subtilisin, procaine pepsin and Tritrirachium album proteinase K can also be used.

Alternatively, chemical reagents can be used to split the protein into peptides. For example, cyanogen bromide can be used to cleave proteins at methionine residues. Chemical fragmentation can also be applied by limited hydrolysis under acidic conditions. Alternatively, BNPS-skatole can be used to resolve at the tryptophan site. Using chemically or using induced ladder or aminopeptidase treatment with isothiocyanate, it can also be used partly NH ₂ -terminal degradation.

II. Chromatography As used herein, the term “chromatography step” or “chromatography” refers to a method for separating chemical substances and is widely available in the art. The preferred approach uses relative velocities at which chemicals are adsorbed from a moving stream of gas or liquid on a stationary substance, usually a finely divided solid, a sheet of filter material or a thin film of liquid on the surface of the solid. It is done. Chromatography is a versatile method that can separate a mixture of compounds even if there is no prior knowledge of the number, nature or relative amount of the individual substances present. This method is for the separation of biological chemical compounds (eg amino acids, protein fragments, peptides, proteins, phospholipids, steroids, etc.) and volatile aromatic mixtures such as fragrances and flavors and complex mixtures of petroleum. Widely used. The most widely used columnar liquid technology is high performance liquid chromatography in which the pump forces the liquid mobile phase under high pressure into a highly efficient closely packed column. A recent overview of chromatographic techniques is described by Meyer M., 1998, ISBN: 047198373X and Cappiello A. et al. (2001) Mass Spectrom. Rev. 20 (2): 88-104, incorporated herein by reference. Has been. Other recently developed methods described in the art and new chromatographic methods that have become available in the art can be used as well. Some examples of chromatography include reverse phase chromatography (RP), ion exchange chromatography, hydrophobic interaction chromatography, size exclusion chromatography, gel filtration chromatography or affinity chromatography such as immunoaffinity and immobilized metals There is affinity chromatography.

  Chromatography is one of several separation techniques. Electrophoresis methods such as capillary electrophoresis, free flow electrophoresis, etc. and all variants are another member of this group. In the latter case, the driving force is an electric field, which exerts different forces on different ionic charge solutes. Resistance is the viscosity of the solvent that does not flow. The combination of these forces creates a unique ionic float for each solute. Some examples include sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and native gel electrophoresis. Capillary electrophoresis includes capillary gel electrophoresis, capillary zone electrophoresis, capillary electrochromatography, capillary isoelectric point, and affinity electrophoresis. These techniques are described in Mckay P., Introduction to Chemistry, Science Seminar, Genetech Inc., Department of Regenerative Science, incorporated herein by reference.

III. Buffer The method of the present invention analyzes the flagged peptides or identified peptides in the analytical device such as separation conditions in the primary run, reaction conditions in the modification step, separation conditions in the secondary run and mass spectrometer. In order to require compatibility between conditions. As described above, the combination of chromatographic conditions and the chromatographic shift induced by the modification reaction in the primary run and the secondary run, the flagged peptide or identification peptide from each fraction obtained from the protein peptide mixture in the primary run. Determine the likelihood of isolation. Similarly, as described above, in preferred embodiments, the chromatographic conditions for primary and secondary runs are the same or substantially similar.

  In a further preferred embodiment, the buffers and / or solvents used in both chromatographic steps allow an efficient progression of chemical and / or enzymatic reactions in a modification step between the two chromatographic steps. It is compatible with the conditions required to make it. In particularly preferred embodiments, the nature of the solvent and buffer in the primary run, secondary run and modification steps are the same or substantially similar. In a further preferred embodiment, the buffer and solvent are compatible with the conditions required to perform mass spectrometry. To define such buffers and solvents requires adjustments and fine adjustments (and such conditions are not available in the prior art). An example showing this tuning is described in Example 9, for example.

  For some embodiments of the present invention with certain types of flagged or identified peptides, the same or substantially similar buffer that can be used throughout the primary run, modification step, secondary run, and analytical procedure Designing one set of liquids and / or solvents is very difficult if not impossible.

  For example, chemical and / or enzymatic reactions to modify peptides in the modification step require specific reaction conditions that are incompatible with the buffers used in the primary and / or secondary runs. . In these cases, the buffer / solvent conditions within the fraction are changed before and / or after the modification step, such as extraction, lyophilization and redissolution steps, precipitation and redissolution steps, appropriate buffer / solvent steps. It is carried out using methods described in the art such as dialysis against solvents or even rapid reverse phase separation with steep gradients.

  Another troublesome issue may be the buffer / solvent composition present in the protein peptide mixture prior to initiating the primary run. Application of the pretreatment steps referred to above in this application may require specific buffer / solvent conditions that are incompatible with the buffer / solvent for performing the primary run. Alternatively, conditions for the preparation / isolation of proteins from that biological source can result in contamination of the protein mixture or protein peptide mixture with compounds that negatively interfere with the primary run. is there. Under these circumstances, the buffer / solvent composition of the protein mixture or protein peptide mixture is changed to be compatible with the primary run. Such modifications include methods described in the art such as extraction, lyophilization and reconstitution steps, precipitation and reconstitution steps, dialysis against appropriate buffers / solvents or even steep rapid reverse phase separations. It is implemented using.

  In yet another embodiment of the present invention, the secondary run buffer / solvent is not compatible with performing flagged or identified peptide analysis to elute. In such a case, the buffer / solvent in the fraction recovered from the secondary run is changed to make the conditions compatible with, for example, mass spectrometry analysis. Such modifications include methods described in the art such as extraction, lyophilization and reconstitution steps, precipitation and reconstitution steps, dialysis against appropriate buffers / solvents or even steep rapid reverse phase separations. It is implemented using. Alternatively, fractions with flagged or identified peptides are collected and recombined for a third series of separations, hereinafter referred to as tertiary runs. This tertiary run is designed so that the eluting flagged or identified peptide can be analyzed with a mass spectrometer. Examples of strategies and pool strategies are described in Example 18, for example.

Equivalents Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. For example, chromatography can in many cases be replaced by electrophoretic methods. Electrophoresis techniques include (capillary) gel electrophoresis, (capillary) electrochromatography, (capillary) isoelectric point and affinity electrophoresis. In yet another equivalent example, the modification can also be a physical modification. For example, exposure of the peptides to high temperatures may result in (partial) denaturation of the thermosensitive peptides, so that these peptides acquire another chromatographic behavior.

  For example, the present invention provides a method for isolating a subset of peptides from a protein peptide mixture, wherein (a) first separating the protein peptide mixture by chromatography into peptide fractions; (b) exposing each fraction to high temperatures. And (c) isolating the physically modified peptide by a second chromatography, wherein the chromatography of the first and second separation steps is performed in the same chromatographic type, Methods are provided wherein the chromatographic conditions are preferably the same or substantially similar. In the reverse mode, peptides that are unmodified after exposure to high temperature are isolated in step c). In certain embodiments, exposure to high temperatures can even be applied during the secondary run rather than before the secondary run.

  Another possibility is that the primary or secondary run is performed in the presence of a magnetic field. This magnetic field then specifically affects the elution or migration of peptides that are sensitive to magnetism. For example, magnetic particles coated with a specific antibody directed against phosphotyrosine can be added to the protein peptide mixture. The phosphotyrosine-containing peptide is specifically affected by the magnetic field.

Example Example 1: Specific chemical alteration protein peptide mixture of methionine residue, produced according to the method described in the present invention, for modification was chosen relatively rare amino acid methionine. As proven in the literature, one approach to modifying methionine is by sulfoxide formation and chemical oxidation that can lead to sulfone formation. Peptides containing methionine can be converted to their sulfone derivatives, for example by using strong oxidation conditions with performic acid or other peracids (Toennies and Homiller, 1942 and Hirs, 1956). Stronger oxidation conditions are rather severe and not sufficiently selective. Formation of methionine-sulfoxide proceeds at the point of contact of methionine and air. However, in the presence of 0.5% H ₂ O ₂ at room temperature and low pH (1% TFA), the reaction is complete in less than 30 minutes. Interestingly, under these mild conditions, two other residues that are very sensitive to oxidation, cysteine and tryptophan, are both little or not oxidized. This conclusion was reached by oxidizing a wide variety of Trp, Cys and Met peptides and then subjecting the reaction products to HPLC analysis and mass spectrometry. An example of the specificity of the reaction is shown in FIG. Both methionine modifications (sulfoxide and sulfone-derivatives) are both more hydrophilic than the unmodified methionine (sulfone derivatives are less severe than sulfoxide derivatives). The specific mild chemical oxidation of peptides containing methionine residues towards methionine-sulfoxide is due to the specificity of modification to methionine and the optimal properties of separating flagged peptides from unmodified peptides This was preferentially used in the present invention. Experiments have shown that this methionine modification can be used efficiently to separate methionine sulfoxide modified peptides from unmodified peptides on a large scale in complex peptide mixtures or protein peptide mixtures under the conditions of the present invention. It has been proven. One important element of the present invention is that the hydrophobicity is greatly reduced when the peptide is converted from methionine to its methionine-sulfoxide embodiment. This is illustrated by a shift in the elution of the oxidized peptide to a lower concentration of organic solvent during reverse phase chromatography (referred to herein as a frontal or hydrophilic shift). While using different methionine-containing peptides, it is demonstrated that reverse phase chromatography conditions can be used to efficiently separate a wide range of peptides containing oxidized methionine from a pool of unmodified peptides. It has been demonstrated that depending on the chromatographic conditions, the hydrophilic shift in the elution of oxidized peptides can be significantly different. The results show that by using the correct conditions, a shift from 3 minutes to more than 7 minutes in the standard gradient for lower modifier concentrations can be obtained. This is illustrated for one peptide run in different systems (Table III). Large shifts were systematically observed in the NH ₄ Ac / methanol system. Smaller but still significant shifts were observed with TFA / acetonitrile or HCOOH / acetonitrile combinations. In principle, all systems listed in Table III can be used in the sorting process. In all the examples below, we used 0.1% TFA / acetonilittle or 0.1% HCOOH / acetonilittle mixture. It should be clarified that the system for sorting peptides does not exclude the use of other solvent systems. The HCOOH / acetonitrile combination is one approach that can be used if the flagged peptide is to be analyzed by electrospray MS. Interestingly, the hydrophilicity shift of flagged peptides (even if derived from the same fraction within a primary chromatographic run) is not identical and can even vary significantly. Oxidation thereby appears to have a variable effect that may depend on the sequence. More specifically, the methionine peptide that has been recovered at 1 minute intervals in the primary run is then eluted as its sulfoxide form within a larger time interval (eg within 4 minutes) in the secondary run. Become. This is a major advantage because the selected methionine-containing peptide elutes in the secondary run at larger time intervals, which significantly increases the resolution capacity of the separation system. As a result, the co-elution of the flagged peptide is reduced and the peptide elutes more gradually and with a lower degree of compression, thereby allowing a better presentation for identification to the mass spectrometer.

  An alternative approach to alter the methionine side chain within the peptide is reaction with alkyl halides such as methyl iodide resulting in the formation of sulfonium ions (Rothgeb et al., 1977). This reaction proceeds slowly and is complete after more than 8 hours. A protein peptide mixture is produced according to the method described in the present invention. For example, a primary run is performed on an anion exchange column and fractions are collected. This fraction is modified with, for example, methyl iodide, which reacts specifically with peptides containing methionine residues. As a result, the peptide containing methionine is modified (the methionine residue is modified to its sulfonium ion) and the resulting flagged peptide is not loaded on the ion exchange column. It moves differently than the modified peptide. The chromatographic conditions for the primary run and secondary run are performed under the same or similar chromatographic conditions. More specifically, as a result of this modification, the elution rate of the flagged peptide is increased when the flagged peptide is passed over an anion exchanger (eg MONOQ or DEAE-column), and the cation exchanger The elution rate decreases on (eg MonoS, phosphocellulose).

Example 2: Specific chemical modification of cysteine residues A protein peptide mixture is generated using one of the methods described hereinabove and specific chemical modification of cysteine residues is performed. This modification is based, for example, on the specific conversion of a cysteine peptide into a more hydrophilic derivative that undergoes a hydrophilic shift during reverse phase HPLC. Several reagents can meet these requirements. For example, reactions with iodoacetamide, iodoacetate, ethyleneimine, promoethylamine, acrylamide and 4-vinylpyricin all convert cysteine to a compound that behaves more hydrophilic under reverse phase conditions. Furthermore, all these compounds undergo oxidation by H ₂ O ₂ resulting in the formation of their corresponding sulfoxide derivatives which are even more hydrophilic. It is important to mention here that the shift due to oxidation is not as pronounced here as in the case of methionine oxidation. However, when the shift between the free thiol cysteine derivative and its modified oxidized counterpart was combined, an overall shift of the flagged peptide similar to that measured for methionine sulfoxide formation was observed (Fig. 4). The following reaction scheme (i) shows how a cysteine residue can be specifically chemically modified in such a way that modifications can be used to separate flagged cys peptides from unmodified peptides. An example of whether it can be modified is shown. Such a protocol is as follows. The protein mixture is dissolved in 8M urea in 1% TFA and first treated with H ₂ O ₂ (final concentration 1%) for 30 minutes at 25 ° C., resulting in all the presence in the protein mixture This leads to sulfoxide formation of methionine residues. The protein is then precipitated overnight at −20 ° C. after the addition of 4 volumes of ethanol. The precipitated protein is recovered by centrifugation and washed once with 1 ml of ethanol-water (3: 1 volume ratio). The washed protein pellet is redissolved in 8M urea, 0.1M Tris-HCl pH 8.6, and a 2-fold molar excess of tributylphosphine is added to convert all SS bridges to thiol groups. Converted. Peptides are generated by specific resolution (most suitably trypsin is used), the protein peptide mixture is separated by RP-HPLC (primary run), and the non-removed fraction in each collected fraction during the secondary run. Collect the fraction so that the flagged peptide can be separated from the modified peptide. In each fraction, the cysteine residue is converted to its S-propionamide derivative by reaction with acrylamide in pH 8.6 buffer (Sechi and Chait, 1998). Immediately following this reaction, oxidation is performed with H ₂ O ₂ in 1% TFA to convert the S-propionamide derivative to the more hydrophilic sulfoxide mode. The latter is described in (i) below.

  Both reactions reach completion in a short time and no intermediate product can be detected (FIG. 4). Furthermore, these reactions can be carried out sequentially without removing reagents in intermediate steps. Thereby, the entire mixture obtained after the last oxidation step was run on an RP-HPLC column and flagged from the unmodified peptide by using the same or very similar chromatographic conditions as during the primary run. Peptides can be separated. Thereafter, the flagged peptide is transferred to an analyzer such as a mass spectrometer to determine the identity of the flagged peptide and its corresponding protein. This procedure is repeated for each fraction collected during the primary run. In order to optimize the time required for chromatographic separation and analysis, a peptide sorter consisting of multiple columns in parallel and / or in series can be used.

In one variation of the reaction sequence, the protein pre-oxidation and precipitation steps can be omitted, starting with protein resolution to produce a protein peptide mixture. A first oxidation step is then performed on the protein peptide mixture at acidic pH, followed by reduction with excess NaBH ₄ at pH 8.6, followed by other modification steps (reaction with acrylamide and oxidation) as described above. It is. All of these reactions can also be carried out continuously without intermediate purification steps.

  Yet another alternative method for selecting for cysteine-containing peptides involving a one-step procedure is to convert the SH-containing peptide into its mixed disulfide embodiment (5,5'-dithiobis (2-nitrobenzoate) or DTNB). Based on the reaction with.

  The mixed disulfide aspect of the peptide is more hydrophobic than its SH counterpart and elutes later in the peptide sorting process. This method also allows discrimination between free SH and disulfide peptides. In fact, by omitting the reduction step, in the present invention, only peptides carrying free SH are isolated, whereas SS peptides are not isolated. However, if the protein or peptide mixture is reduced prior to the first run of the sorting process, the sum of SH and SS peptides is sorted.

Example 3: Specific chemical modification of the sum of methionine and cysteine residues The procedure includes a procedure for specifically modifying cysteine residues, except that the pre-oxidation step of methionine residues is omitted. Are identical (see Example 2). The reaction sequence begins with the reduction of the protein mixture with tributylphosphine followed by enzymatic or chemical resolution. Protein peptide mixture is separated by RP-HPLC, each fraction is altered by reaction with acrylamide, it is oxidized with H ₂ O ₂ immediately thereafter. Here the methionine-peptide is oxidized with the modified cysteine peptide and both types of flagged peptides show a hydrophilic shift when chromatographically separated using conditions similar to the primary run. FIG. 5 summarizes the various reaction sequences in Met-sorting, Cys-sorting and Met + Cys-sorting modes.

Example 4: Specific Modification of Phosphoserine and Phosphothreonine-Peptides A protein peptide mixture is produced as described above in this application, and a class of peptides modified simultaneously with or after translation is specifically isolated. Here, an example of a strategy for isolating phosphoserine and phosphotrenion-containing peptides is provided. Phosphoserine and phosphothreonine-containing peptides are modified to their dehydroalanine and dehydroamino-2-butyric acid derivatives, respectively, by alkaline β-elimination of the phosphate moiety. Michael addition of ethanethiol converts the former into S-ethylcysteine derivatives and the latter into β-methyl S-ethylcysteine derivatives (Weckwerth et al., 2000). These thioether-containing amino acid derivatives are modified into their respective sulfoxide embodiments after reaction with H ₂ O ₂ similar to the oxidation of methionine residues. In order to avoid mixing with methionine-peptide and to avoid β-elimination at cysteine during alkaline treatment, the protein mixture is first oxidized with performic acid according to Hirs (1956). This step converts methionine to the sulfone manner and cysteine residues to cysteic acid. After dialysis against purified water, the protein mixture is digested with trypsin in 50 mM ammonium bicarbonate at 37 ° C. overnight at a ratio of 1: 100 trypsin to total protein. Trypsin digest (10 μl) is added to 50 μl of a 2: 2: 1: 0.65 mixture of H ₂ O / DMSO / EtOH / 5M NaOH, and 60 μl of ethanethiol is added. The reaction mixture is heated at 50 ° C. for 3 hours and after cooling, quenched by adding 60 μl of 20% acetic acid and 10 μl of acetonitrile. The protein peptide mixture is separated by reverse phase chromatography (primary run) at a fraction number that allows the flagged peptide to be separated from the unmodified peptide in each collected fraction in a secondary run. In each fraction, the peptide is oxidized with H ₂ O ₂ . Since methionine and cysteine were oxidized in the early oxidation step, here only S-ethylcysteine and β-methyl-S-ethylcysteine are oxidized to their sulfoxide derivatives (see Schemes iii and iv). ). These sulfoxide derivatives are much more hydrophilic. Each fraction was run on an RP-HPLC column and the same or similar chromatographic conditions as in the primary run were used to flag the peptide (S-ethylcysteine sulfoxide and phosphoserine and phosphothreonine-containing peptides, respectively). β-methyl-S-ethylcysteine sulfoxide) is separated from the unmodified peptide. Thereafter, the flagged peptide is transferred to an analyzer such as a mass spectrometer to identify the corresponding flagged peptide and its phosphorylated protein. Furthermore, a neutral loss scan of RSOH (where R = ethyl) (78 amu) during mass spectrometry allows further confirmation of the authenticity of both types of flagged peptides (Steen and Mann, 2000). The latter fact is attributed to S-ethyl cysteine sulfoxide and β-methyl-S-ethyl cysteine sulfoxide representing phosphoserine and phosphothreonine-containing peptides, respectively, due to the neutral loss found after measurement of the flagged peptide by mass spectrometry. This means that there is an internal control for the authenticity of.

That the alkaline β-elimination reaction can be carried out under milder alkaline conditions with 0.5 M LiOH at 4 ° C., thereby replacing the mixture of H ₂ O / DMSO / EtOH / 5 M NaOH described above. It is advantageous to point out (Sakaguchi et al., 2001).

An alternative system for sorting phospho-peptides exploits the hydrophobic difference between phosphorylated and dephosphorylated peptides by reverse phase chromatography at pH 5.0. The procedure outlined below is an example of such an approach. A protein peptide mixture is produced as described above in this application. Here, peptides present in the mixture are separated by RP-HPLC using 10 mM NH ₄ Ac, pH 5.0 / acetonitrile (or NH ₄ AC / methanol or other) as an elution solvent, and a fraction of 1 minute is obtained. Collect with. Peptides present in these fractions are treated with common phosphatases (eg alkaline phosphatase). A dephosphorylated peptide is not as hydrophobic as its phosphorylated precursor, and thus undergoes a hydrophobic shift during secondary chromatographic separation under chromatographic conditions identical or substantially similar to the primary run, Therefore, they can be sorted. Here, the hydrophobicity shift is pH 5.0, which is greater than the lower pH value, making the use of 0.1% TFA or 0.1% HCOOH system in the sorting process less attractive. It is important to mention that.

One interesting option is the use of a separation system specifically adapted for the separation of phosphoryl-containing compounds. Such a system is, for example, an absolute boss combined with 5 mM tetrabutylammonium phosphate at pH 5.0 as solvent A and 60 mM NH ₄ H ₂ PO ₄ and methanol as solvent B (5 mM tetrabutylammonium phosphate). It can be composed of absobosphere nucleoside-nucleoside materials (Alltech).

  Another way to sort phosphopeptides using dephosphorylation methods is based on the loss of negatively charged phosphoryl groups. In this case, the primary and secondary runs are performed on an ion exchange column or by electrophoretic means. For example, when primary and secondary runs are performed on a MonoQ-column or DEAE-column at pH 6.0, all dephosphorylated peptide species have a weaker binding power to the anion exchanger. Therefore, a forward shift is indicated. Dephosphorylated peptide species exhibit an anodic shift and the use of capillary electrophoresis can provide a similar effect and can lead to a sorting process.

  Any sorting procedure based on a dephosphorylation step that can be carried out either by enzymatic (eg general or specific phosphatase) or chemical (eg β-elimination under alkaline conditions) can be used for various phosphorylated It is important to emphasize that it offers the possibility to choose for species.

Yet another way to sort for phosphopeptides is based on the formation of non-covalent complexes between phosphopeptides and Fe ^{3+ -chelates} . A protein peptide mixture is produced as described herein. Peptides present in the mixture are contained in the mixture in primary run 1 by RP-HPLC using 10 mM NH ₄ Ac, pH 5.0 / acetonitrile (or NH ₄ Ac / methanol etc.) as the elution solvent. The peptides present are separated and collected at 1 minute time intervals. Peptides present in each of these fractions are collected in the secondary run on the same chromatography column, but now in a solvent containing Fe ³⁺ and iminodiacetate that forms a dichelate complex with the phosphopeptide. To separate. This complex elutes at a different position compared to that of the free phosphopeptide, allowing isolation of the phosphopeptide. This differential chromatography again forms the platform for an efficient sorting process.

Example 5: Selection for ε-N-acetylated peptides Acetylation of a certain number of lysine ε-amino groups and possibly other factors of nucleosome histones H2A, H2B, H3 and H4 modifies the chromatin structure and transcription Leading to increased activity. Altering the degree of acetylation is likely to be associated with cell proliferation and may indicate apoptosis, necrosis or some other pathological situation. Furthermore, the acetylated state makes a very early difference between already normal cells and neoplastic cells (eg prostate cancer). Again, the present invention can be used to selectively sort on acetylated peptides, for example using deacetylation as a shifting principle for peptide sorting. An example of such a strategy is provided below for illustrative purposes. A protein peptide mixture from the nuclear extract is generated by trypsin resolution of the isolated protein. Trypsin splits with Arg and Lys but not with acetylated lysine side chains. The resulting peptide mixture was used with 0.1% TFA as solvent A and 0.09% TFA in 70% acetonitrile as solvent B with an increasing gradient of 1% solvent B / min and 80 μl / Separation in Run 1 by RP-HPLC at a flow rate of minutes (column inner diameter 2.1 mm, length 250 mm). Eluting peptides are collected at 1 minute intervals. All fractions are dried, redissolved in an appropriate buffer and treated with histone deacetylase (HDA). For example, fully or partially purified preparations of yeast Rpd3 (class 1), yeast HDA1 (class II) or NAD ⁺ dependent Sir2 class proteins (for review see Furmai et al. 2001 thing). Due to deacetylation, the peptide becomes more hydrophilic and elutes at lower acetonitrile concentrations. Due to this difference in hydrophilicity and by applying the present invention, it is possible to separate deacetylated peptides (flagged peptides) from unmodified peptides during the secondary run. The elution shift is comparable to the shift measured for the modification from methionine to methionine sulfoxide peptide. The nature of each flagged peptide and its corresponding protein is determined, for example, using MS / MS or by measuring the mass of each flagged peptide very accurately. This allows identification of proteins in the original protein mixture.

To quantitatively determine the ε-N-acetylated peptide difference between two samples (eg different cell types), either H ₂ ¹⁶ O (sample 1) or H ₂ ¹⁸ O (sample 2) Protein peptide mixture is produced by trypsin digestion. Both protein peptide mixtures are mixed prior to the primary run and further processed as described above. The flagged peptide from any random protein X in sample 1 co-elutes in the secondary run with the same flagged peptide from the same protein X in sample 2. Since the flagged peptides from Sample 1 and Sample 2 carry ¹⁶ O and ¹⁸ O, respectively, they appear as two peaks in mass spectrometry. Calculate the intensity or surface area of the peak and the ratio of the peptide flagged with twice ¹⁶ O to the flagged peptide with twice ¹⁸ O is the degree of acetylation of this peptide in both comparative samples. Is directly proportional to

Example 6a: Selection of NH ₂ -Terminal Peptides Derived from NH ₂ End-Blocked Proteins In Vivo Another application of the invention is the isolation of peptide subsets derived from NH ₂ -end-blocked proteins It is in. Most of these can be acetylated peptides (eukaryotes) or formylated peptides (prokaryotes). In order to select only NH ₂ terminal blocked peptides and avoid loss of amino terminal blocked peptides containing lysine residues, samples containing proteins are pre-treated. In one approach, the sample is first guanidized with O-methylisourea at pH 10 to convert the lysine side chain to its guanidinium derivative. The α-amino group reacts with this reagent much more slowly than the ε-amino group (Plapp et al., 1971) and is therefore completely or minimally converted to its guanidinium derivative.

According to the present invention, the protein is then subjected to trypsin digestion. Trypsin cleaves both arginine and homoarginine, albeit at a slower rate (the latter is derived from guanylated lysine), so digests were generated except those containing a blocked protein amino terminus. All peptides produce a free α-amino group. The protein-peptide mixture is then passed over a reverse phase column and, in each of the collected fractions, a fraction that makes it possible to separate the modified peptide from the unmodified peptide during the secondary run. Separated by number. For example, in each fraction is added phenyl isocyanate (PIC) that reacts with the free NH ₂ groups of the peptide. As a result, all peptides with free NH ₂ groups acquire phenylcarbamoyl (PC) groups, making the peptides more hydrophobic (FIG. 6). NH ₂ - peptides derived from endblocked protein is not altered. The peptide mixture is applied to an RP-HPLC column and the modified peptide is separated from the unmodified identified peptide (ie peptide blocked at the amino terminus) by using similar or identical chromatographic conditions as in Run 1. Thereby, in this example, most peptides are modified and delayed (hydrophobic shift), while a subset of unmodified peptides elute at unchanged positions during the secondary run. This is called the “reverse sorting procedure”.

The range of hydrophobic shift can be modified by changing the chemical nature of the NH ₂ -terminal reactive derivative. Methods known in the art describe various isocyanate (IC) modification reactions that can be used as an alternative to PIC in the sorting process described in this chapter. For example, reaction with trifluoroacetyl-IC, allyl-IC, naphthalene-IC, fluorescein-IC, etc. can be used as well. The hydrophobic shift of a peptide with a free α-NH ₂ -group can also be obtained by any other quantitative modification reaction specific for the α-NH ₂ -group. The reagent list includes, for example, acetyl-N-hydroxysuccinimide and all acylating reagents, F-moc-N-hydroxysuccinimide, trinitrobenzene sulfonic acid (TNBS) or nicotinoyl (oxy) succinimide. In the final selection of reagents and conditions, the modification reaction is limited to α-NH ₂ -groups only, with at least 90%, preferably 95%, more preferably 99% and most preferably peptides with free NH ₂ groups. It will be clear that will further modify the ratio.

Example 6b: Selection This example NH ₂ -terminal peptides derived from proteins with a free NH ₂ terminus, the NH ₂ -terminal peptides derived from proteins with a free NH ₂ -terminal present in the protein peptide mixture how to Indicates whether sorting is possible. A particular advantage of this method is due to the fact that it is possible to obtain a peptide flagged with its sulfonic acid group attached to its NH ₂ terminus that is ideally suited for high-throughput MALDI-PSD analysis (Keough T et al (1999)). Samples containing protein are first treated with tributylphosphine followed by iodoacetamide in protein denaturing buffer. This step leads to derivatization of the cysteine side chain, followed immediately by a guanidination reaction that converts lysine to homoarginine. The α-NH ₂ group is then blocked with an isothiocyanate derivative such as phenyl isothiocyanate (PITC) or the well-known soluble Braunitzer reagent (1,5-disulfonylnaphthalene-3-isothiocyanate). Thereby, the protein present in the mixture is then derivatized at its SH group (as an acetamide derivative), its ε-NH ₂ group (as homoarginine) and its α-NH ₂ group (as its thiocarbamoyl derivative). Has been. Subsequent splitting with trypsin now generates a new free α-NH ₂ group set at each new split site. These can be efficiently blocked by reaction with trinitrobenzene sulfonic acid (TNBS). The final pretreated protein peptide mixture is now composed of four types of NH ₂ end-blocked peptides. First, peptides derived from in vivo blocked proteins; ie α-NH ₂ acetylated (eukaryotic) or formylated (prokaryotic) peptides; second, peptides blocked with TNBS; A peptide blocked with pyroglutamic acid in 3 and can occur spontaneously after trypsin resolution before a glutamine residue; and fourth a peptide blocked with a thiocarbamoyl (TC) derivative. The latter represents a peptide subset corresponding to the protein amino terminal peptide. Of the four types of NH ₂ terminal blocked peptides, only the TC peptide is known to be sensitive to acid treatment and will release the NH ₂ terminal residue according to well-known Edman chemistry. Thereby, treatment of the peptide mixture with a selected from the group consisting of enriched TFA removes the first amino acid of the TC-peptide that produces a new free NH ₂ terminus. At this point, the peptide mixture is separated and recovered in Run 1 in each of the recovered fractions in a fraction number that allows separation of the modified peptide from the unmodified peptide during the secondary run. The In each fraction in is added NH _2-specific reagent, selectively alter the peptide subsets free NH ₂ group. Such a reagent may be TNBS or an acetylated compound that leads to a more hydrophobic peptide. However, in certain embodiments, the reagent may be composed of chemistry developed by Keough T. et al., 1999 or similar compounds that modify peptides having a sulfonic acid moiety in the α-NH ₂ group. These flagged peptides can again be selectively sorted using RP-chromatography or ion exchange chromatography procedures performed in accordance with the present invention. One important aspect of peptides bearing NH ₂ -terminal sulfonic acid groups allows for very fast and easy deduction of amino acid sequences, and therefore efficient MALDI-based analysis of sorted peptides. And their specific fragmentation under the conditions currently used in MALDI-TOF-MS mode, paving the way for identification.

An example of a continuous chemical or enzymatic process leading to the sorting of easily sequenceable peptides derived from proteins with free NH ₂ termini is summarized below:
Step 1: Reduction and subsequent reaction with iodoacetamide Step 2: Conversion of lysine side chain to homoarginine Step 3: Conversion of free α-NH ₂ group in thiocarbamoyl derivative Step 4: Precipitation of modified protein Or purified by gel filtration.
Step 5: Divide with trypsin.
Step 6: Modify the peptide with trinitrobenzene sulfonate.
Step 7: Treat with concentrated TFA.
Step 8: Dilute with water and separate the protein peptide mixture in the primary run.
Step 9: Modify peptides in each fraction with NH ₂ end blocking reagent, preferably by sulfoacetylation.
Step 10: Sort peptides flagged in secondary run.
Step 11: Analyze the flagged peptide by MALDI-PSD or ESI-CID or MALDI-CID.
Step 12: Identify peptides based on the sequence produced by a series of y ions.

Since the protein always leads to a new NH ₂ terminus, which generally does not carry any known blocking group, this procedure can be specially adapted to study the internal resolution of proteins (Example 8). Similarly, this sorting process again performs positive selection for the sulfoacetylated peptide, and even with the unmodified peptide even if the sulfoacetylation reaction (here alternating reaction) did not proceed completely It is also meaningful and valuable to emphasize that this is a direct approach to avoiding or minimizing contamination.

Example 7: Technical represented by one identification peptide one protein of a complex mixture such as a selected cell lysates of the induced NH ₂ -terminal peptides from proteins present in complex protein mixture (NH ₂ terminus peptide) , Hereinafter referred to as individual peptide mass-based proteomics (IPMBP, see Example 10). This procedure begins with the conversion of the protein cysteine with iodoacetamide or a similar SH-specific reagent known in the art. The protein is then reacted with O-methylisourea to convert ε-lysine to its guanidinium derivative (homoarginine). It is important to point out that while the α-NH ₂ group of the protein does not change under the reaction conditions used, the ε-NH ₂ group changes. In the next step, the protein is acetylated, for example with acetyl-N-hydroxysuccinide. In the next step, a protein peptide mixture is produced, for example by trypsin resolution, and this protein peptide mixture is separated in a first chromatography step. For each fraction, the free NH on Peptide ₂ - adding groups and quantitatively react trinitrobenzene sulfonic acid (TNBS). It is important to note that peptides derived from the amino terminus of a protein cannot react with this reagent because its NH ₂ group was previously blocked with an acetyl group. Peptides with free NH ₂ groups acquire trinitrobenzene groups (TNB), making these peptides more hydrophobic. Thus, when the peptides from each fraction are separated on the RP-HPLC column run under chromatographic conditions similar to those in Run 1, the modified TNB-containing peptides are unmodified identified peptides (ie all blocked at the amino terminus). Peptide). In this setup, the isolated unmodified identification peptide will be derived from the amino terminus of the protein and will contain an NH ₂ -terminal acetyl group (eg, when a eukaryotic cell extract is used).

In another method, the protein mixture is pretreated by converting protein cysteine to its carboxamide derivative. In the next step, the protein is acetylated with acetyl-N-hydroxysuccinide at both its ε-NH ₂ and α-NH ₂ groups. A protein peptide mixture is then generated by trypsin resolution. Since all lysine side chains have been previously acetylated, trypsin resolution is preferentially at the COOH terminus of arginine. All additional steps including the peptide sorting process are performed as described above. This leads to the isolation of the amino terminal peptide of all proteins present in the mixture. These are sorted as unmodified identification peptides.

In an alternative approach, the protein is acetylated with an equimolar mixture of acetyl- and trideuteroacetyl-N-hydroxysuccinide that leads to differential isotope labeling of the free α-NH ₂ -terminus of the protein. In the next step, the protein mixture is digested with trypsin and the protein peptide mixture is passed through a reverse phase column so that the modified peptide can be separated from the unmodified peptide in run 2 in each of the collected fractions. The number of fractions is separated. To each fraction is added trinitrobenzene sulfonic acid (TNBS) that reacts quantitatively with free NH ₂ groups on the peptide. Thus, again, once the peptides from each fraction were separated on the RP-HPLC column run under chromatographic conditions similar to those in Run 1, the modified TNB-containing peptides were isolated from the unmodified identified peptides. To be separated. In this setup, peptides derived from proteins with a free α-NH ₂ group (modified during our procedure) are now labeled with a double-tagged CH ₃ —CO / CD ₃ —CO— moiety. and What to, peptides derived from already vivo blocked proteins, carrying a CH ₃ -CO group. Unmodified identified peptides from each fraction are transferred to a mass spectrometer to determine the mass and sequence of each individual peptide. Importantly, this analysis has already blocked these peptides in vivo since a group of peptides derived from proteins with free α-NH ₂ groups appears as doublets (separated at 3 amu). It is possible to distinguish peptides derived from proteins.

Alternatively, TNBS is replaced in the process with phenyl isocyanate (PIC) or similar compounds that can block free NH ₂ groups. In the case of peptides formylated at the NH ₂ terminus, the same sorting procedure is followed. Thereby the formylated peptide, a double CH ₃ -CO / CD ₃ -CO tags to sort with labeling peptides (see above). It is important to note that our sorting procedure also sorts peptides bearing pyroglutamic acid at its NH ₂ terminus. Such peptides can be formed during enzymatic resolution when NH ₂ -terminal glutamine is produced. Mass spectrometry fragmentation of peptides is used, it can identify the NH ₂ -terminal acetyl and pyroglutamic acid so as to identify the NH ₂ -terminal peptide and any internally generated peptide.

Example 8: Identification of proteins that have undergone internal proteolytic processing and localization of processing sites within whole cell lysates using the present invention Often, proteins are internally internalized due to the action of specific protheases. Divided. This phenomenon is seen, for example, at the onset of apoptosis due to caspase activation. Internal protein processing can also be an important step during normal cell development, and such a process can play an important physiological role. Furthermore, protein splitting within the precursor molecule is a process that leads to protein maturation. The detection of these processes is a fundamental element in modern proteomics. Our invention allows us to identify both the nature of the processed protein and the location of the processing site. A typical but non-limiting experimental protocol is described below. First, proteins derived from whole cell lysates are reduced with tri-butylphosphine and the SH groups are blocked with iodoacetate. The reaction is carried out at a pH of 8.6 and a denaturing concentration of guanidinium-HCl (6M). For reverse sorting methods (and thus unmodified peptides are selected as identified peptides) it is recommended not to use urea-containing buffers. In fact, prolonged contact with urea leads to carbamylation of the peptide, which will be sorted as an undesirable product as well. In this step, excess reagent and buffer are removed by precipitation in 4 volumes of ethanol at −20 ° C. overnight. The protein precipitate is collected by centrifugation and redissolved in a small volume of 6M guanidinium in pH 8.5 phosphate buffer. Alternatively, the reagents and buffer can be removed by a gel filtration step in 6M guanidinium-HCl in phosphate buffer at pH 8.5. To convert the free NH ₂ group to its corresponding acetyl or nicotinoyl derivative, acetyl or nicotinoyl N-hydroxysuccinimide ester is added. Alternatively, as in Example 7, a 1/1 mixture of acetyl and trideuteroacetyl derivatives is used. In this second example, a 1/1 mixture of H4 or D4-type nicotinoyl derivatives is utilized (Munchback, M et al., 2000). The acetylation reaction is terminated by adding 1 molar excess of pH 8.5 Tris-HCl to the acetylating reagent and diluted to 1 M guanidinium-HCl or to 0.5% NH ₄ HCO ₃ . Dialyze and then digest with trypsin. The resulting peptide is subjected to primary chromatographic separation. Each fraction is then treated with a reagent that reacts quantitatively with the newly generated free peptide α-NH ₂ — group (eg, trinitrobenzene sulfonate, acetyl N-succinimide ester, phenyl isocyanate, etc.). A rerun of these treated fractions in the secondary run will then translocate all the peptides reacted in the last reaction step towards a more hydrophobic position, while already blocked in vivo or primary All peptides that are NH ₂ end-blocked by pre-treatment prior to the run or peptides with NH ₂ -terminal pyrrolidone carboxylic acid are recovered as unmodified identification peptides at the same position they eluted during the primary run. Peptides derived from the newly generated NH ₂ terminus that provide information about both the nature of the processed protein and the exact cleavage site by comparing the peptide patterns of protein lysates from two different samples Can be identified. The above experiments can be varied in a number of ways while still retaining the general principle of sorting identified peptides. For example, after converting the protein with SH-reactive and NH ₂ -reactive compounds, the protein is digested with trypsin in H ₂ ¹⁶ O (sample 1) and H ₂ ¹⁸ O (sample 2). Using this experiment, differential quantification of the range of protein processing can be studied between two samples. Thereby, after trypsin resolution, sample 1 and sample 2 are combined in equal proportions and this mixture is separated in the first chromatographic run. Each fraction is then treated with a reagent that reacts with free α-NH ₂ groups (eg, trinitrobenzene sulfonate, acetyl N-succinimide ester, phenyl isocyanate, etc.). The rerun of these treated fractions in the secondary run then translocates all peptides that reacted in the last reaction step towards a more hydrophobic position, while already blocked in vivo or primary All peptides that are NH ₂ end-blocked by pre-treatment of the run or peptides with NH ₂ -terminal pyrrolidone carboxylic acid are recovered as unmodified identification peptides at the same position they eluted during the primary run. Light ⁽¹⁶ O) and heavy ⁽¹⁸ O) peptides are chemically very similar, each peptide pair was separated in the same way, also ionize in the same way. During mass spectrometry, light and heavy peptides separate from the heavy peptide having a mass increase of 4 amu. This separation is sufficient to accurately measure the differential quantification of the range of protein processing in the two samples.

Example 9: Buffer System The key elements of the invention are 1) the reaction conditions used to modify the peptide, and 2) the mass spectrometry used to analyze and identify the flagged or identified peptide The type of approach, in relation to and in the selection of these integrated peptide separation conditions. For the purpose of illustrating this point, the following describes some examples of how to select a methionine-peptide from a protein peptide mixture taking into account this integral aspect of the procedure. In one example, a primary run is performed in a TFA / acetonitrile system and the oxidation step is performed in 1% TFA / H ₂ O ₂ . Secondary runs are similarly performed in the TFA / acetrinitrile system, while peptide mass measurements are performed by PSD-MALDI-RETOF-MS or MALDI-TOF-MS which are insensitive to trace amounts of TFA (see below). Thus, in this protocol, the counter ion TFA does not change throughout the procedure from the beginning of the primary run to the entire secondary run. When identification of flagged or identified peptides is performed by electrospray-ionization (ESI-MS), it is known that TFA forms ion clusters and interferes with MS-measurements (Mirza and Chait, 1994), TFA-system is not recommended. Thus, as a second example, since the HCOOH / acetonitrile system allows efficient ionization by ESI, both primary and secondary runs are now performed in this system. However, in the presence of HCOOH, it is not possible to carry out an intermediate oxidation step to produce a sulfoxide-methionine peptide because it leads to the formation of performic acid and thus the transformation of the methionine side chain in both sulfoxide and sulfone derivatives. Thereby, here the oxidation step is carried out in a mixture of 1% TFA and 0.5% H ₂ O ₂ . In this case, the nature of the counterion is not the same between the two successive chromatographic steps and the modification, which may affect the ion pairing effect between the secondary runs. Here, the disturbance effect is not important due to the relatively low concentration of TFA. This can be a problem when the TFA concentration is increased or when counterions are used with stronger ion-pairing effects during the modification process. In summary, it is preferred to keep the buffer properties unchanged throughout the primary and secondary runs, during the modification process and during mass spectrometry. If this is not possible, or if the buffer in the chromatography process is different from the solvent used in mass spectrometry, a third chromatography run can be performed.

  In the same way, it may be important to take into account the buffer ions present in the protein peptide mixture before starting the primary run. Ideally, the buffer ions in the protein peptide mixture should be the same as those used during the primary and secondary runs. If the buffer ions in the protein peptide mixture are too divergent, the required adaptation can be obtained in several ways available in the art. For example, this can be obtained by dialyzing the protein mixture against an appropriate buffer prior to trypsin digestion. Alternatively, a short reverse phase (RP) -separation with a steep slope can be added before starting the primary run. During this fast RP separation, the salt is removed and the peptide acquires the correct counterion that will be used in the primary and secondary runs. The peptides eluting from this high speed RP step are combined, lyophilized and dissolved in a buffer suitable for the primary run. The procedure that brings the peptide mixture under most ideal ionic conditions is referred to herein as the conditioning step. One example where conditioning of the peptide mixture is important is described below.

  To inhibit the closing, human plasma is prepared by adding citrate buffer. If tryptic digestion of such a whole plasma protein preparation is directly subjected to the primary chromatography step, the peptide separation will be affected by the citrate originally present in the peptide mixture. If the unmodified peptide is now transferred for a secondary run with little citrate present, there may be an undesirable shift due to changes in ion pairing. This type of shift is greater for the more hydrophilic peptides eluting at the beginning of the gradient than for the slower eluting hydrophobic peptides. The effect of citrate in the primary run is first avoided by passing the protein peptide mixture through a high speed RP column using a steep gradient of organic solvent. All peptides eluting over the full gradient are collected and dried by lyophilization or vacuum dried and redissolved in an appropriate buffer prior to the primary run. Conditioning the protein peptide mixture ensures the same or identical chromatographic conditions throughout the sorting procedure. The conditioning process is also important as a cleaning process to remove compounds that can progressively contaminate the sorting column.

Example 10: Individual peptide mass-based proteomics (IPMBP)
According to the present invention, it is possible to select a flagged peptide or a subset of identified peptides from a complex peptide mixture or a protein peptide mixture. Further in accordance with the present invention, peptides and corresponding proteins are identified with a suitable analyzer such as a mass spectrometer. A MALDI-TOF mass spectrometer is used to measure the mass of the peptide, which is not always sufficient to unambiguously identify the peptide and its corresponding protein. In this example, several approaches are described for increasing the information content of an isolated flagged or identified peptide. This allows an increasing number of the peptides to be determined unambiguously by simple determination of their mass using a mass spectrometer. This approach is called individual peptide mass-based proteomics (IPMBP).

10.1 IPMBP for peptides produced with endoproteinase-LysC
By using the present invention, it is possible to select a peptide containing one or more analytes of specific amino acids. In order to increase the number of peptides that can be clearly identified, the knowledge that this amino acid should be present in the selected peptide is used. One approach consists in building a sub-database containing only the mass of peptides known to contain at least one residue of a specific amino acid. For example, if methionine is selected as the specific amino acid, a sub-database with the mass of peptides containing at least one methionine is created, and the mass of each flagged peptide containing methionine is compared to this database. To be screened.

  A further increase in the proportion of identified peptides flagged as clearly determined can be obtained by using specific proteases. In one example, endoproteinase-LysC is used. In this example, a database containing all peptides that would be derived by in-protein endoproteinase-Lys-C digestion of human and E. coli proteins (extracted from SwissProt database release 39.0). Was built. From this database, a sub-database of peptides is created that meets the specific criteria that its monoisotopic mass must be between 700 Da and 4000 Da and must contain at least one methionine residue. It was. This sub-database is indexed according to increasing peptide mass and can then be used as a unique identifier for its parent protein, ie its mass measured to exactly 3 digits corresponds to a unique peptide sequence The number of running peptides was calculated. From these calculations, it was observed that 91% of the calculated human peptide mass and 95% of the calculated E. coli peptide mass served as unique immobilization peptides (FIG. 7). Similarly, the number of proteins in the database that contained at least one of these unique identifiers was calculated, and it was observed that over 80% of the proteins for both species could be identified in this way. It was. In order to use this strategy for high-throughput peptide-based proteomics, it is necessary to measure peptide mass with very high accuracy. As recently published, such high mass accuracy is well within reach of, for example, a Fourier Transform Mass Spectrometer (FTMS) using an internal calibration procedure (O'connor and Costello, 2000). If this level of accuracy is not reached, a very rapid drop in identification power can be expected immediately. Similarly, from a statistical point of view, the larger the database, the less obvious assignments are produced than the smaller one. Therefore, it is preferable to direct the IPMBP-search algorithm to a single species or organism. For these simulated experiments, an endoproteinase Lys-C was used that produced, on average, a larger peptide compared to trypsin or chymotrypsin digestion. Use of the latter enzyme or a combination of different proteases results in a peptide database with more inputs, thereby reducing both the number of proteins that can be uniquely identified by IPMBP and the number of unique peptide masses. become.

10.2 Enhancement of peptide information content To obtain more stringent standards without using time-consuming MS / MS analysis, the flagged peptide information content is N-hydroxysuccinimide ester and N-hydroxy trideuteroacetate. succinimide free NH in the peptide using an equimolar mixture of the ester ₂ - further enhanced by changing the basis of the specific. As a result of this conversion reaction, the flagged or identified peptides acquire a predetermined number of CH ₃ —CO (CD ₃ —CO) groups depending on the number of free NH ₂ groups in these peptides. To do. The number of groups acquired can be easily deduced from the range of observed mass shifts of peptide doublets. For example, the shift of 3amu corresponds with the presence of one NH ₂ group, 3 and the shift of 6amu corresponds with two NH ₂ groups, 3, 6 and shift 9amu has three NH ₂ groups in the peptide The presence of Alteration of the free NH ₂ group is most suitably performed after protein digestion but before the start of the primary run. Acetylation of free NH ₂ groups within the peptide increases the hydrophobicity of the peptide. Despite this effect, for example, the range of hydrophilic shifts (δmin and δmax) obtained after methionine oxidation (see Example 1) is similar to that when the peptide was not acetylated. Thereby, the present invention is equally applicable with this approach. Using this approach in combination with the approach described above, the following information is obtained for each flagged peptide: (1) mass determined by MS, (2) specifically selected amino acids (eg, Number of residues of (methionine) and (3) number of free amino groups. This combined information significantly increases the number of flagged peptides or identified peptides that can be clearly identified by screening databases and sub-databases as described above.

  Furthermore, this approach can be used to determine the proportion of peptides present in the two mixtures. In this example, the peptide from one sample is acetylated with acetic acid N-hydroxysuccinimide ester and the peptide from the second sample is acetylated with trideuteroacetic acid N-hydroxysuccinimide ester. The ratio of the two isotopic forms of each flagged peptide measured in the mass spectrum is then used to make a quantitative comparison. In differential quantitative methods, O-methylisourea is used to determine the number of lysine residues in the tryptic peptide, and this additional information improves the overall success of protein identification using conventional search methods. A similar approach was recently published by Brancia et al. (2001) who showed that. The combination of this approach and the present invention further significantly improves the proportion of peptides that can be clearly identified.

  Given a certain mass within the separation system (eg during the primary run) because it allows to identify multiple peptides with the same or very similar mass but different hydrophobicity or net charge Peptide elution or migration time is an important additional information.

10.3 IPMBP by selecting for NH ₂ -terminal tryptic peptides
In this invention, for example, it is possible to sort on NH ₂ terminal peptides of NH ₂ end blocked proteins (Example 6a), but this idea extends to NH ₂ terminal peptides of all proteins in the sample (Example 7). FIG. 6 shows a specific sorting method for peptides derived from in vivo NH ₂ blocked proteins (most likely NH ₂ acetylated, NH ₂ formylated or pyroglutamylated). ing. Using a variation of this procedure (Example 7), we can also sort specifically on most if not all of the NH ₂ -terminal peptides, if not all of the proteins present in the protein peptide mixture. This procedure also allows discrimination to be made between peptides derived from NH ₂ end-blocked proteins in vivo and from proteins with free NH ₂ groups. Finally, the mass of these sorted NH ₂ -terminal peptides can be easily determined using, for example, a mass spectrometer.

An important advantage of this approach is that it selects for the amino terminal peptide of the protein. As a result, identification of the protein corresponding to a peptide is greatly simplified because the search for correlating peptide mass with peptide mass stored in the database can now be limited to the mass of the amino-terminal peptide in the database. Has been. As a result, for most peptides it is possible to unambiguously correlate the peptide with its corresponding protein. In an ideal situation, all NH ₂ -terminal peptides can be considered as single representative identifying peptides of their corresponding parent proteins, and the problem of protein identification is mainly related to 1 protein-1 peptide correlation. I can return. This means that for a mixture of 1000 different proteins, 1000 different identified peptides must be searched. Since the extent of processing at the NH ₂ terminus during protein maturation in vivo is not always known, it is somewhat necessary to confirm this assumption by simple computer simulations using genomic DNA sequences. Accompanied by difficulties. For example, β-cytoplasmic actin is first synthesized as Met-Cys-Asp-Asp-Asp-Ile-, but eventually acetylated with the addition of acetyl groups after successive removal of Met and Cys. -Asp-Asp-Asp-Ile ... (Redman and Rubenstein, 1984). The problem of “unpredictable” NH ₂ -terminal protein processing is solved by first selecting and then identifying all identified peptides by MS / MS or PSD approaches. These studies indicate that the sorted NH ₂ -terminal peptide contains either arginine or homoarginine (hArg), which ionizes very efficiently and predominantly converts y-type fragment ions during MS / MS analysis. It is not overly complex because it has been found to lead to a spectrum that can be generated and easily interpreted (Biemann, 1990). As already mentioned in Section 10.2, the elution or migration time of the identified peptide can be a valuable and sufficient additional parameter to be combined with its total mass to fully identify the identified peptide. Thereby, the mass of all identified peptides combined with their chromatographic properties along with information on which protein this specific peptide is derived from is stored in a relational database. This means that in most cases the mass of the identified peptide can be unambiguously correlated with its parent protein.

10.4 Use of IPMBP in a Quantitative Differential Proteome Approach The procedure for using IPMBP in a quantitative proteome approach with an identified peptide consists of the following steps: ie, according to the procedure described in Example 7, protein peptide The protein from Mixture 1 is first modified with cysteine, guanidated and then N-terminal acetylated. The protein is then digested with trypsin in the presence of H ₂ ¹⁶ O. The same procedure is performed for protein peptide mixture 2, but this time a trypsin digest is performed in the presence of H ₂ ¹⁸ O. Trypsin not only acts as a catalyst for splitting its target peptide bond, but also incorporates two oxygen atoms derived from water at the split site (see, eg, Schnoelzer et al., 1996) (Rose et al ., 1983). The peptide derived from protein peptide mixture 1 thereby is labeled at the COOH terminus with two ¹⁶ O-isotopes, while the peptide from protein peptide mixture 2 now has two ¹⁸ O-isotopes. The same peptides derived from different mixtures are distinguished by 4 amu. Here, the peptide mixtures are combined and passed over the first column (Run 1). The peptide is recovered again in the form of fractions and labeled with a specific reagent bearing a hydrophobic (or hydrophilic) group at its α-amino group. At this time, peptides derived from NH ₂ end-blocked proteins (in vivo or in vitro) do not migrate in the secondary run and can be recovered at the same elution time interval as run 1. In contrast, all modified peptides that reacted in the NH ₂ group after the primary run now undergo a hydrophobic / hydrophilic shift and separate from the position they were taken before they were labeled. To do. When a hydrophilic reagent is used to modify the free α-amino group, a hydrophilic shift of the modified peptide is observed compared to the NH ₂ end-blocked peptide. However, since the free α-amino group of the peptide is already hydrophilic, most blocking reagents lead to more hydrophobic compounds that elute later than the free amino group peptide. At the time of mass spectrometric analysis of the isolated identified peptide, it is then separated by two types of peptide doublets or 4 amu (difference between having two ¹⁶ O and two ¹⁸ O isotopes) and blocking in vivo Those derived from the purified protein are detected. The ratio of peak intensity or peak surface area reflects the relative ratio of the corresponding proteins in the two mixtures. The second type of doublet incremented by 7 amu (the difference between having two ¹⁶ O and two ¹⁸ O isotopes, the difference between having three H atoms and three D atoms. 1) derived from the protein in the sample that had been separated and had a free NH ₂ terminus. The ratio of peak intensity or peak surface area again reflects the relative ratio of the corresponding proteins in the two mixtures. The reaction scheme for the quantitative differential NH ₂ -terminal peptide approach is summarized in FIG.

An alternative to the ¹⁶ O / ¹⁸ O differential labeling method is a chemically synthesized, containing at least one deuterated (or any type of heavy isotope ¹³ C, ¹⁵ N) amino acid, The use of flagged peptides or identified peptides that allow sufficient separation by mass spectrometry of identified peptides versus “heavy” synthetic identified peptides. Here, the “heavy” peptide serves as an internal standard. Thereby, synthetic peptides are added to the protein peptide mixture in known quantities and sorted along with their natural counterparts. Comparison of peptide peak ratios in a mass spectrometer allows a relative quantitative estimate of naturally identified peptides versus added synthetic peptides.

Such isotopically labeled flagged or identified peptides may contain, for example, deuterated leucine (eg, L-leucine-d ₁₀ which produces a mass difference of ₁₀ amu) or deuterated methionine. The latter may be convenient when Met-containing peptides are sorted (see, eg, Examples 1, 18, 19 and 20).

Because most of the tryptic peptides are terminated with arginine or lysine, and chemical peptide synthesis starts at the COOH-terminus and proceeds toward the NH ₂ terminus, either deuterated lysine or deuterated arginine Starting from this, all peptides can be synthesized, while other amino acids can be attached as their natural derivatives. In this case, it is possible to use a solid support on which deuterated lysine or deuterated arginine is already linked by a resolvable linker arm. Such solid phase resins can be used as general starting materials from which all types of heavy flagged peptides or identified peptides can be synthesized therefrom by conventional solid phase peptide synthesis.

Example 11: Peptide sorter apparatus using a single column system The basic protocol of the present invention for isolating, for example, a methionine-containing peptide from a protein peptide mixture consists of two successive chromatographic steps. That is, one is carried out in a solvent system that has been found to produce the most suitable shift between oxidized methionine-peptide and unmodified peptide, and is most likely with either electrospray or MALDI-ionization procedures. Compatible RP-HPLC process. The second RP-HPLC run performed after the oxidation step is performed under the same or very similar chromatographic conditions, so that only the oxidized peptide is shifted forward, while the unmodified peptide is originally The elution time will remain. This principle is used in several ways to separate methionine peptides from non-methionine peptides. Thus, in the single column system schematically represented in FIG. 9, the primary run (Run 1) of the whole cell peptide mixture is transferred to a standard reverse phase column (eg C-18RP—with an internal diameter of 2.1 mm × 25 cm). Column, Vydac Separations Group, CA). This is called the primary run and the UV absorbance profile of the peptide from the E. coli lysate is shown in FIG. After an initial homogeneous wash step with 5% solvent B for 10 minutes, the column is eluted with an acetonitrile gradient that increases linearly with 1% solvent B per minute for 95 minutes. Buffer A consists of 0.1% trifluoroacetic acid, while Buffer B consists of 0.09% TFA in 70% acetonitrile. The flow rate is kept constant at a rate of 80 μl / min and 80 μl fraction is collected in a 0.5 ml Eppendorf tube. Collect a total of 40 fractions. The first fraction collected is called fraction nr10 (which elutes between 18 and 19 minutes after the start of the gradient and corresponds to 23% solvent B). The last collected fraction (which elutes between 57 and 58 minutes after the start of the gradient and corresponds to 63% solvent B) is given the number 49. In this experimental setup, peptides that contain oxidized methionine residues that are specifically chemically modified only in the methionine residues present in the peptides in the fraction are referred to herein as Met-SO peptides. Specific modification is performed as follows. Each fraction was vacuum dried and consisted of 1% TFA in water, to which 30% H ₂ O ₂ stock solution was added to give a final concentration of 0.5% H ₂ O ₂ . Re-dissolve in. Oxidation is allowed to proceed for 30 minutes at 30 ° C., after which the solution is immediately loaded onto the RP-column for the secondary run. According to the present invention, it is highly preferred to rerun all fractions after oxidation on the same column under the same or similar chromatographic conditions. Using the same conditions as in Run 2, the Met-SO peptide elutes between 6 and 2 minutes before the bulk of the unmodified peptide. One approach is to run all fractions separately and do this for each of the 40 fractions. Such an approach not only requires a large amount of HPLC time, but also occupies significant working time on the mass spectrometer. In order to use both HPLC instruments and mass spectrometers more economically and to accelerate the entire process, we pool several fractions of the primary run and move forward with the unmodified peptide from the previous fraction. The number of separations was reduced by avoiding overlapping of peptides flagged for shifting, and avoiding overlapping of flagged peptides from one fraction and flagged peptides from the other fraction. Before setting up this protocol, we measured the hydrophilicity shift of a significant number of synthetic methionine-containing peptides after oxidation, with most shifts being less than 6% and more than 2% solvent B, thereby It was recognized that the gradient used in the experiment was within a 6-2 minute window (see also Table III). In the two-minute zone that exists between the shifted peptide and the bulk of the peptide material, we found very few methionine-peptides, but instead, the few unmodified peptides that spread from the large peak of the bulk peptide. Already detected. In order to avoid overlap in the elution of flagged peptides coming from two fractions in this particular experimental setting, it is necessary that there is an elution difference of at least 6 minutes between these two fractions (eg , For fraction 10, this would have been fraction 17). However, for safety reasons, we added an additional 5 minutes elution time after elution of the large peak fraction before allowing the elution of the next group of oxidized methionine-peptides. This means that for the secondary run we were able to mix the fractions separated in the primary run by a 12 minute interval. Thereby we combined fractions 22, 34 and 46 with fraction 10. Similarly, we combined fractions 11 with fractions 23, 35 and 47 etc. A complete list of fraction pool operations for secondary runs is shown in Table IVA. The UV absorbance profile of a standard secondary run (run 2A combining fractions 10, 22, 34 and 46) is shown in FIG. Methionine SO-peptide was collected each time within an interval of 6 to 2 minutes before the time when the unmodified peptide was expected to elute. Thereby, in the run 2A example above, the oxidized peptide was recovered in fractions 4-7, 16-19, 28-31 and 40-43 (Table IVA). Oxidized peptides in the remaining secondary runs were recovered as further listed in Table IVA. In total, therefore, we must perform 12 such secondary separations in succession to cover all fractions from the primary run. It should be added here that the time interval and window utilized in the sorting process described herein can be varied depending on the selected chromatography system or the type of component to be sorted. For example, in Example 18, we describe a sorting process using different time intervals and windows to sort methionine peptides as well, but using the HCOOH / acetonilittle system. Each particular problems temporal intervals and windows (e.g. phospho - peptide sorting, sorting of N-.epsilon.-acetylated peptides, NH ₂ - terminus such sorting peptides) because must be adapted to the self-evident That is.

Met-SO peptides eluting during the secondary run can be passed directly into the ion source of an on-line connected mass spectrometer (eg ESI-based mass spectrometer) or further MALDI-TOF / RETOF- It can also be collected in small aliquots for MS analysis or spotted directly in the form of small droplets on a MALDI-target plate for high-throughput MALDI-MS analysis. Alternatively, the sorted Met-SO peptides can be collected in an Eppendorf tube and recombined for a possible third series of separations (referred to herein as tertiary runs). The latter may be necessary when peptide sorting is performed in a TFA-containing system, while the analysis is performed by ESI-MS. In fact, TFA is known to cause ion clustering during electrospray and significantly impair peptide detection and MS / MS analysis (Mirza and Chait. 1994). This is not the case when either 0.05% HCOOH or pH 5.7 10 mM NH ₄ AC buffer is used as the counter ion. That the use of the latter system can produce a shift in peptide elution time when compared to the TFA-system used in the previous run, and in some cases can lead to unwanted peak accumulation and thus inefficient peptide identification. Should be recognized. In Tables IVB and IVC, we present two different schemes that illustrate how fractions derived from secondary runs can be pooled towards performing a tertiary run. If the same counter ion is used in the solvent throughout the different runs, the fractions that elute one after another can be combined. For example, run 2A fraction 4-7 is run 2E fraction 8-11, run 2I fraction 12-15, run 2A fraction 16-19, run 2E fraction 20-23, run 2I fraction 24-27, Run 2A fractions 28-31, run 2E fractions 32-35, run 2I fractions 36-39, run 2A fractions 40-43 (marked in blue in Table IVB) can be combined. The remaining fractions are combined in a similar manner as shown in Table IVB, leading to four pools whose components can be separated into four tertiary runs: 3A, 3B, 3C and 3D. When using 0.05% HCOOH in a tertiary run, it is recommended to mix only half of the fraction each time. Thereby, for run 3'A, run 2A fraction 4-7 is now run 2I fraction 12-15, run 2E fraction 20-23, run 2A fraction 28-31 and run 2I fraction 36-39. Pool with. Other combinations are again listed in Table IVC and separated into 8 different tertiary runs (3'A to 3'H). Still other combinations of pooling fractions from secondary runs to perform tertiary runs are possible. Tertiary runs as described above are important to better disperse the peptides over several runs, but it is more efficient and faster to identify them as soon as the peptides elute the column during the secondary run. That is. From a time perspective, the latter is not optimal for a single column peptide sorting apparatus because the Met-SO peptide elutes at intervals separated by an 8-minute block where no recovery is possible. These 8-minute blocks can be filled with two 4-minute eluates when three columns are run simultaneously. The design of such a three-column peptide sorter is described in Example 12.

  Reverse sorting in which the modified peptides that form the majority of the peptides of the original protein-peptide mixture are discarded or used for other analysis, while unmodified peptides are sorted and recovered as identified peptides If a process is used, the following procedure is obvious.

Assuming that all the modified peptides shift between 6 and 2 minutes before the elution position of the identified peptide, assuming that the 1st fraction was taken (W ₁ = 1 minute) Using peptide shift values similar to those used in the methionine oxidation example used in Example 1, modified peptides eluting between -6 and -2 minutes are not analyzed, but when taken in the primary run The identified peptide is recovered within the same time interval. Here it should be clear that the unmodified peptide represents a small fraction of the original mixture, while the modified peptide forms the bulk of the peptide.

Similarly, it may be pointed out that due to the absence of large amounts of peptide in the secondary run, the window through which the unmodified peptide elutes during the secondary run may be somewhat wider. is important. Thus, unmodified identified peptides are better recovered within a slightly wider window than W ₁ , eg, 0.5 minutes before and after the W ₁ time interval.

  Similar to the example of methionine oxidation, here too we can mix fractions 10, 22, 34 and 46 of the primary run. Here, the modified peptides eluting in fractions 4-7, 16-19, 28-31 and 40-43 are discarded, while the identified peptides are now fractions 9.5 to 11.5 minutes, 21. Recovered in 5-23.5 minutes, 33.5-35.5 minutes and 45.5-47.5 minutes.

  Thereby, the reverse sorting process can be performed on the same equipment as the normal sorting process with minimal changes in the peptide recovery program.

  Again, it should be apparent to those skilled in the art that the shift time will vary depending on the chromatography system and conditions and the modified chemistry or procedure used.

Example 12 Apparatus for Sorting Three Column Peptides The procedure using Example 11 based on a single RP-HPLC column for all steps to reduce overall peptide sorting time is This is done using three columns operating synchronously. A schematic diagram of such a sorting system is shown in FIG. This peptide sorting apparatus contains three identical RP columns that are run under exactly the same conditions (flow rate, gradient, etc.). In order to achieve the same conditions, each of these columns is in communication with a high pressure pump and a solvent mixer to precisely control the flow rate and gradient within the three columns (FIG. 12A). Alternatively, three columns are fed by a single high pressure pump, while the flow rate for each column is monitored by a splitter valve that can be flow controlled (FIG. 12B). On column I, fractions 10, 22, 34 and 46 from run 1 are applied. Exactly the same flow rate and gradient as run 1 is created. The column is first washed with 0.1% TFA in 5% solvent B for 10 minutes (eg, 70% acetonitrile in 0.09% TFA). The gradient as in Run 1 is then continued with a gradient of 1% solvent B per minute. From the 4th minute to the end of the 7th minute (fractions 4-7), the Met-SO peptides are recovered or they are directed into the ion source of the MS-device for analysis. Alternatively, 4-7 eluates are collected in small aliquots using, for example, a Micro Blotter (Applied Biosystems, Foster City, CA, USA) for further analysis by MALDI-TOF-MS. At 8-15 minutes, the eluate is again directed to waste. At 16 minutes, a second boost of Met-SO peptide from fraction 22 in run 1 is recovered. This collection and analysis takes place between 16-19 minutes (fractions 16-19). The eluate is then discarded again until the 28-31 minute interval, during which time a third boost of Met-SO peptide originally derived from fraction 34 is collected. Collection is stopped at the 32nd minute, and the eluate is further introduced into the waste. The gradient is continued with additional recovery of fractions 40-43, as in Run 1, and completed 58 minutes after the start of the gradient, followed by a 30 minute re-equilibration step with 0.1% TFA Continue. At this time, the column II into which the fractions 14, 26 and 38 are charged is run under exactly the same conditions as described above for the column I. Here, the Met-SO peptide is stored at time intervals of 8-11, 20-23, and 32-35. Column III is subjected to fractions 18, 30 and 42 and run under the same conditions and periodicity as columns I and II. Here the Met-SO peptide elutes at time intervals of 12-15, 24-27 and 36-39. When runs on three columns are operating simultaneously, there is no longer any dead interval between Met-SO peptide analyses. In fact, as demonstrated in Table V, we reduced the product of column I between 8-11, 20-23 and 4-7, 16-19, 28-31 and 40-43 minutes. The product of column II is analyzed between 32 and 35 minutes, while that of column III is stored for the time of 12-15, 24-27 and 36-39 minutes. These ten time intervals can be perfectly aligned, resulting in a continuous flow of Met-SO peptide to the MS instrument. Again, the methionine peptide originally captured in a total of 10 minutes during the primary run is now sent out after sorting into the MS instrument spread over the total 40-minute time frame, creating much better analytical conditions. It is important to mention that. In addition, the flow rate can be reduced during the time interval during which the Met-SO peptide elutes so that the mass spectrometer can select and analyze the eluted peptides more efficiently. Thereby we can use a kind of peak parking (Davis et al, 1995) procedure when the Met-SO peptide elutes the sorting system. This requires adaptation of the elution time in other connected columns. Using combined fractions 11, 23, 35 and 47 separated on column I, fractions 15, 27 and 39 on column II, and fractions 19, 31 and 43 on column III, this time, the second A triple run is performed. Again, the Met-SO peptides are 5-8, 17-20, 29-32 and 41-44 time intervals for column I, 9-12, 21-24 and 33-36 for column II. Recovered at intervals, and for columns III at intervals of 13-16, 25-28 and 37-40. All combinations of Run 1 fractions and preservation of Met-SO peptides are represented schematically in Table V. Valve operation throughout the entire procedure is depicted in Table VI. Thereby, by using a three-column peptide sorter, we can now separate all Met-SO peptides from the complex mixture in a total of four secondary runs. Since all runs will take about 120 minutes including loading, washing, elution and re-equilibration, the entire Met-SO sorting process from whole cell lysate can be accomplished in approximately 500 minutes. This sorting process can be monitored directly using an on-line connected mass spectrometer. Alternatively, the eluate of the Met-SO peptide can be collected for further combination and analysis in a tertiary run, otherwise on a MALDI-target that allows continuous high-throughput analysis, The eluate can also be spotted into small aliquots. In addition to the RP-HPLC chromatography conditions used here, the same sorting process can be carried out with a column system that allows a much faster elution time, thereby reducing the sorting process overall. Should also be mentioned.

Example 13: 9-column peptide sorting apparatus FIG. 13 shows the configuration of a 9-column peptide sorting apparatus. The first three columns are connected to one gradient pump and one sample injector. There are second and third series of columns, each connected with one gradient pump and one sample injector. Nine columns, which can be small disposable columns, are divided into three units. Unit A contains columns I, II and III, while units B and C contain columns I ′, II ′, III ′ and I ″, II ″, III ″, respectively. Of fraction II, fraction 24 on column II and fraction 36 on column III.Each charge procedure is followed by a washing step with solvent A for at least 10 minutes, after which the gradient is started. (Increase of 1% solvent B per minute) The gradient is initially passed only over column I. This column is preconditioned between 0 and 5 minutes. Elutes between -9 minutes and the column is washed between 10-16 minutes At 17 minutes the valve is placed in such a way that the gradient passes through column II which is first equilibrated for 1 minute. The Met-SO peptide is 18 Recovered in 21 minutes and the column is washed to 22-28 minutes At 29 minutes, the gradient is directed into column III, which is preconditioned for 1 minute.The Met-SO peptide is 30-33. Elution between minutes followed by a wash from 34 minutes to the end of the gradient System B (columns I ', II' and III ') is charged with fractions 13, 25 and 37 respectively. Developed using the same program, with a delay of 3 minutes relative to A. Thereby, the Met-SO peptides are 10-13 (I '), 22-23 (II') and 34-37 (III ' ) For system C (columns I ″, II ″ and III ″), the same program is used in fractions 14, 26 and 37 with a delay of 6 minutes for system A. Corresponding Met-SO peptides are recovered at intervals 14-17 (I "), 26-29 (II") and 38-41 (III "). Thus, in the I-step procedure, nine fractions at a time The Met-SO peptides are sorted from the four additional such runs in which the fractions are loaded and recovered as shown in Table VII, all the Met-SO peptides present in the original mixture. Leads to complete sorting of peptides, Table VIII provides a complete description of valve settings during complete runs An important aspect of the 9 column peptide sorting system is the overall design of the system And that the column dimensions are different from those used in the primary run, which results in different elution times even when the RP-adsorbent and solvent system are kept the same. There is a possibility, which means that. One solution for this problem is the use of colored are added to the peptide mixture (Ala) _n -Arg synthetic mixture (see Example 14). In this way it is possible to use a continuous colored peak as a reference point leading to fraction collection in the primary run, thereby collecting the fraction between or around two consecutive colored peaks. it can be. then the same colored reference components can also be used to direct a fraction collector to the peptide sorter. use of 9 column type peptide sorter in combination with reference compounds, sorting for the NH ₂ -terminal peptide Particularly useful in the process (see Examples 6 and 7), the latter process does not alter the sorted peptides and Stay with criteria mixture while the majority of peptides that are not sorted is delayed, it is separated from the colored reference mixture.

  One run with this 9 column sorter is achieved within about 60 minutes, which means that the complete sorting process is completed within ± 300 minutes or 5 hours. It is important to mention here that small and inexpensive columns can be used in this sorter. Since only one fraction is processed per column, flow accuracy is not as important as in systems where multiple fractions are loaded on a single column. All runs can be performed at relatively low pressures as well, with less demand on the pumps and valves. The 9 column sorter is also better adapted for situations where the hydrophilic shift is greater or less than that regularly measured during methionine sulfoxide formation. This is the case when a methanol chromatography system is used or when cysteine derivatives are oxidized (see Table III). The different peptide sorters described here are limited examples that do not exclude the construction of similar sorters with different numbers of columns.

Example 14: Calibration of a peptide sorter The efficiency and accuracy of a peptide sorter depends mainly on the reproducibility of the column separation. It is therefore practical to use component mixtures or peptide calibration mixtures that cover the entire solvent gradient range and can be monitored by mass spectrometry, absorbance or other means for regular monitoring of the sorter as well as installation It is. Examples without limiting sense, we here, COOH-terminal arginine residues consisting of several alanine varying bound to chemically synthesized peptide mixture (n is in the range of up to 7-42 Ala _n -Arg) is used. This mixture is synthesized using conventional solid phase synthesis procedures (Merrifield, 1963). Using a mixture of 97% Fmoc-Ala and 3% tBoc-Ala for peptide extension, a premature stop is generated after each cycle in 3% of the growing peptide chain. This type of synthetic strategy produces a mixture in which all components are different from other components by the addition of one alanine that produces a set of peptides that show a continuous change in both mass (71 amu) and hydrophobicity. Thereby, in the case of the above example, a given elution window (W1) can likewise be characterized by one or more peptides from this mixture with a well-defined mass value. Illustrative, non-limiting sense, it is shown elution profiles for _{C18-RP- NH 2 -Ala n -Arg} -COOH mixture on column using as modifier acetonitrile with 0.1% TFA as ion (FIG. 14A). A colored version of such a calibration compound, epsilon referred Ala _n -Lys-Gly-Arg - to permit covalent bond colored section through an amino group, a poly -Ala- peptides with additional lysine residue Can be obtained by the synthesis of

  This peptide calibration mixture is used to monitor the characteristics and characteristics of each column in the peptide sorter apparatus and calibrate the entire system. This can be done by mixing the calibration peptide with a peptide mixture derived from the sample during the primary run. Any adjustment to the elution profile of the calibration compound may include changing the modifier concentration, changing the elution gradient, changing the column temperature, adding an ion pairing agent such as octylamine or dodecyl sulfate or adding tetrahydrofuran or propanol to solvent B, etc. This is done by conventional means well known in the art. The same peptide calibration mixture is also used to calibrate mass spectrometers, especially MALDI-TOF-MS machines, to reach high accuracy.

Example 15: Peptide Identification and Parent Protein Assignment Flagged peptides or identified peptides eluting in a secondary run or from a tertiary column system are passed directly into the ion source of an electrospray mass spectrometer, then Further fragment in MS / MS mode. Partial sequence information is collected from the MS / MS fragmentation spectrum and used for peptide identification in the sequence database. Since peptides that have been flagged in Run 2 over a wide time interval are eluted progressively, the co-elution of these peptides is minimal and the resolution of MS / MS is significantly enhanced (eg, 18). The present invention is also used to identify peptides by MALDI-TOF-MS. In fact, high-throughput MALDI-TOF-MS technology is utilized to rapidly scan flagged or identified peptides. To this end, we have for example a peptide-bead enrichment method (Gavaert et al. 1997 and Gevaert et al. al., 1998). The information obtained is limited to total peptide mass, which is not always sufficient for unambiguous identification if it cannot be measured accurately. There are several ways to recover additional information that leads to a more definitive identification.

For example, for a Met-SO peptide, it can be ascertained whether the peptide contains Met-SO. These peptides are characterized by the efficient neutral loss of methanesulfenic acid (64 amu) observed during mass spectrometry (FIG. 15). After methanesulfenic acid loss, the peptide appears to have lost its vibrational energy, giving it apparent stability in MALDI-PSD analysis. Therefore, it is almost impossible to generate interpretable post-source decomposition (PSD) spectra from Met-SO peptides, thus losing tools for peptide identification using MALDI-mass spectrometry. There are several ways to avoid the PSD problem. First, by treating them with a reducing agent such as N- (methyl) mercaptoacetamide, the Met-SO peptide can be back reduced to its original structure (Houghten and Li, 1981). Second, and as a more convenient method, the Met-SO peptide is converted to its corresponding sulfone derivative using longer incubation with performic acid (Hirs, 1956) or H ₂ O ₂ (eg, 24 hours at room temperature). And further oxidize. Both methionine sulfones and methionine-peptides produce MALDI-PSD decomposition spectra that are much better than the corresponding sulfoxides. It should be noted that at this step, little neutral loss is observed for the sulfone-peptide, producing a better PSD spectrum.

  In an alternative approach, peptides can be fragmented by collision activated dissociation (CAD). This type of fragmentation is relatively less likely to generate sulfoxide-induced dipoles and is therefore an important tool in generating sequence information from Met-SO peptides (Example 18).

Another way to obtain further information is to use partial NH ₂ by using chemically induced ladders with isocyanates (Chait et al., 1993) or aminopeptidases (Caprioli and Fan, 1986). It is based on terminal degradation. This method provides sufficient information at the NH ₂ terminus of all peptides leading to complete identification. Such aminopeptidase digestion or ladder sequencing is particularly beneficial in high-throughput systems, but can only be performed on relatively low-complexity peptide mixtures.

Thereby, the assignment of one or more methionine residues combined with partial sequence information from the NH ₂ terminus of the peptide, the combination of precisely measured peptide masses is not all from the total lysate. Are sufficiently restrictive to unambiguously identify most peptides.

  See also the additional methods described in this step for the identification of peptides and parent proteins based on precisely measured masses at this step.

Example 16: Determination of quantitative relative peptide amounts in two samples To compare protein expression levels in two cell sets or more generally two different samples in a relative quantitative manner, respectively The protein lysate of the preparation is digested with trypsin. In one sample, trypsin digestion is performed in H ₂ ¹⁶ O, while digestion of the second sample proceeds in H ₂ ¹⁸ O. Trypsin has the potential to incorporate two oxygens of the water molecule at the COOH terminus of the newly generated site (Rose et al., 1983 and Schnoelzer et al., 1996). Thereby all peptides of sample 1 trypsinized in H ₂ ¹⁶ O have normal mass, while sample 2 has masses of 2 and 4 amu corresponding to the uptake of one and two ¹⁸ O-isotopes. Contains peptides with an increase (except for most COOH-terminal peptides). The relative ratio of the two ¹⁸ O-embodiments of the peptide depends on several factors including the nature of the peptide, the activity of the enzyme, and the purity of ¹⁸ O-water, and thus ¹⁸ O-uptake for relative quantitative measurements. If is used, the extent of incorporation of two ¹⁸ O-isotopes within the peptide must be considered in the overall calculation (Stewart et al., 2001).

Although digestion is performed separately, all subsequent processing, including methionine-peptide sorting, can proceed on the mixture of the two digests without significant reverse exchange of enzyme atoms. ^{The 16} O- and ¹⁸ O-digests are mixed and subjected to isolation of a modified flagged peptide (Met-SO peptide) on methionine. Peptides with a methionine flag can be identified, for example, as described in Example 15. Light ⁽¹⁶ O) and heavy ⁽¹⁸ O) peptides are chemically and very similar to, each pair will be separated in the same manner. They likewise ionize in the same way. Only during mass spectrometry, they separate into light and heavy peptides. (The latter has a higher mass of 2 and 4 amu due to the incorporation of one and two ¹⁸ O-atoms). Ion separation induced by ¹⁸ O incorporation is sufficient to accurately measure the ratio of light peptide to heavy peptide and thereby determine the ratio of proteins in the two samples (eg, Mirgorodskaya et al., 2000 ). A schematic presentation of the entire procedure is given in FIG. 16A.

To test ¹⁸ O-uptake for relative quantitative analysis, we used one in “normal” ¹⁶ O-water and once in ¹⁸ O-water (purity 95%, ARC Laboratories, Amsterdam, The Netherlands), digested the platelet cytosol and membrane skeletal fraction (prepared as in Examples 20 and 21) using trypsin for 16 hours at 37 ° C. Prior to the primary run, 1 part of ¹⁶ O-digested material is mixed with 2 parts of ¹⁸ O-digested product, and the peptide acidified to 1% TFA and methionine flagged is used to sort the single column peptide. The system was used to sort from the peptide mixture as described in Example 1 (Example 11).

In LC-MS analysis, the ¹⁸ O / ¹⁶ O ratio of peptide ions observed as described above was calculated. The results of this analysis are shown in FIG. 16BB, confirming that the peptide ratio generally fluctuates around 2, and that this type of isotope labeling technique is suitable for quantitative proteome analysis Represents.

We further added ¹⁸ O-labeling by digesting two equal amounts of bovine serum albumin with trypsin, one in normal H ₂ O and the other in H ₂ ¹⁸ O (95% ¹⁸ O). Confirmed the use of the date. After 18 hours of digestion, both peptide mixtures were mixed, separated by HPLC and analyzed by MALDI-TOF-MS. For 19 peptides, we compared the peak heights of isotopes (eg, ¹³ C-isotopes) that were not affected by the labeling procedure. Values were further corrected for the presence of 95% H ₂ ¹⁸ O and combined in FIG. An average ratio of 1.03 was measured for 19 peptides, which corresponds very well to the molar ratio at which the protein digest was mixed at the start of the experiment. Most values agree very well with the predicted values, with extreme values of 0.84 and 1.20 (Table IX). This experiment illustrates that stable isotope labeling during trypsin digestion forms the basis for quantitative differential proteomic studies and is used in the present invention.

Differential isotope labeling can be performed by alternative methods as well, some of which are briefly mentioned below. The labeling procedure is based on known chemical reactions and can be performed at either the protein or peptide level. The following describes a fixed number of reactions used for differential labeling. Peptides can for example be reagent pairs ie methyl isocyanate / trideuteromethyl isocyanate (v); ethyl isocyanate / pentadeutero-ethyl isocyanate (vi); phenyl isocyanate / pentadeutero-phenyl isocyanate (vii); acetyl-N-hydroxy It can be changed with succinimide / trideuteroacetyl-N-hydroxysuccinimide (viii). All of these compounds are known to react specifically and quantitatively with α-NH ₂ and ε-NH ₂ groups. The final choice of modifying reagent will depend on the availability, cost, chemical stability and laboratory comfort of the deuteration aspect of the reagent. Another important aspect is the stability of the adduct during the ionization process within the mass spectrometer. The reaction scheme for each of these reagents in the deuterated embodiment is set forth below.

If a larger mass difference is required between the “light” and “heavy” peptides, use a combination of ¹³ C, ¹⁵ N, and deuterium or a larger deuterated group can do. For example, the use of a hydroxybutyryl group (HO—CD ₂ —CD ₂ —CO—) to specifically label the NH ₂ terminus would allow us to create a difference of 6 amu. . Alternatively, ¹³ CD ₃ for marked label the NH ₂ -terminal - by the use of ¹³ CD ₂ -CO- propionyl group, it will be able to produce a difference of 7 amu. Even more explicitly, through the use of ¹³ C-labeled and deuterated nicotinoyl derivatives (N ¹³ C ₅ D ₄ CO—) we can use a mass difference of 9 amu. Let's go. It should be known to those skilled in the art that N-hydroxysuccinimide or sulfo-N-hydroxysuccinimide ester can be synthesized from all these groups.

Similarly, it is clear that D represents deuterium ² H in the structural formula described in the present invention.

It is also possible to modify the peptide by formation of a Shiff base with deuterated acetol followed by reduction with sodium borohydride (Geoghegan et al., 1979). This reaction has been described to proceed under mild conditions, leading to the addition of only one molecule of acetol per amino group, creating a secondary amine (ix). At this time, the deuterated amine contains 5 non-exchangeable deuterium atoms and will be separated from its undeuterated counterpart by 5 amu. The peptide is modified at both the ε-NH ₂ and α-NH ₂ groups of lysine, leading to a mass increase of 5 amu (for arginine peptides) and 10 amu (for lysine peptides). The underlying reaction is shown below.

  The examples cited herein are merely representative of some of the broader modification reactions that can be used for differential isotope tagging.

Example 17: Quantitative differential proteome display to measure the molar ratio of proteins in a sample The procedure for protein identification via its representative peptide using mass spectrometry is qualitative but not quantitative. Absent. Indeed, peptides exhibit differential loss after their purification, and peptides can be ionized in a highly variable and unpredictable manner depending on their chemical nature and other peptides present in the mixture. This phenomenon is well known as suppression of ionization in the field of MS (Krause et al., 1999). However, as demonstrated in Example 16, mass spectrometry labels one of the two samples with an isotope tag that is chemically indistinguishable but can be distinguished and measured by a mass spectrometer. Quantitative when possible. We now use the same principle of differential isotope labeling to measure the relative ratio of proteins within one single sample. This can be done by adding a known amount of a reference peptide to the sample. These are peptides derived from proteins present in the sample, the sequence of which is sufficient to unambiguously identify the parent protein. The reference peptide is preferably also selected as a readily isolated peptide that also fully ionizes within mass spectrometry. In protocols that select for methionine flagged peptides (Met-SO peptides), the reference peptide is preferably a methionine-containing peptide that also contains an arginine residue or has been processed for efficient ionization. . All proteins to be quantified must be represented by at least one, preferably two or more reference peptides. The reference peptide differs from its synthetic counterpart by differential isotope labeling that is large enough to distinguish both aspects in a conventional mass spectrometer. As pointed out earlier in this application, a difference of 4 amu is sufficient. Such isotope discrimination can be obtained in a variety of ways, and here are some examples. In the most convenient form, the isotope-labeled reference peptide is generated by trypsin digestion of the protein mixture in H ₂ ¹⁸ O. The corresponding synthetic counterpart of the reference peptide is synthesized with its natural isotopes. Such chemical synthesis is carried out on a large scale using a multiple peptide synthesizer (Zuckermann et al., 1992).

An example of a protocol for determining the amount of target protein in a particular sample containing protein is summarized as follows: (i) selecting a reference peptide from the target protein, (ii) corresponding (Iii) digest a protein sample with trypsin in the presence of H ₂ ¹⁸ O, (iv) add a known amount of a synthetic reference peptide to the protein peptide mixture (preferably The amount of the synthetic reference peptide is comparable to the predicted amount of the reference peptide), (v) subjecting the mixture to the present invention to separate the flagged peptide, (vi) the flagged peptide Analyzing with MALDI-TOF-MS, (vii) Reference peptide and synthetic reference peptide are co-eluted in the process and as two peaks in the mass spectrum (Viii) calculate the surface area of each of the two peaks; (ix) the ratio between both peaks is the amount of reference peptide, and correspondingly the amount of target protein in a particular sample. Allow calculation. This protocol can obviously also be used for identifying peptides and can be adapted in several ways. For example, determining the amount of multiple (more than 100) target proteins in a sample and thereby easily extending this to measure the expression level of multiple target proteins in a given sample. it can. Clearly this approach can also be used to measure and compare the amount of target protein in multiple samples. Such results can be used, for example, to predict, monitor or diagnose the effects and side effects of a disease or drug.

In an alternative approach, the synthetic peptide carries a rare isotope, while the reference peptide produced from the protein is a natural isotope. For example, if a methionine-containing peptide is selected, it is possible to incorporate commercially available deuterated methionine (CH ₃ SCD ₂ CD ₂ CH (NH ₂ ) COOH) into the synthetic reference peptide and add 4 amu to the total peptide mass. is there. Alternatively, the synthetic reference peptide also contains deuterated arginine, which in this case adds 7 amu to the total peptide mass. It should be clear that all amino acids from which deuterated ¹⁵ N or ¹³ C embodiments are present can be considered in this protocol. Yet another alternative approach is to design a synthetic reference peptide with a colored fluorescent or otherwise measurable group attached. By introducing a universal color tag that displays the same molecular extinction coefficient for all reference peptides, it becomes easier to quantify the amount of all reference peptides. The quantifiable group is sufficiently stable during normal storage, but should be attached to the peptide with an anchor or linker that is released from the reference peptide by a controlled chemical or enzymatic process. For example, a color dye such as a 2,4,6-trinitrobenzenesulfonate group (maximum molecular absorption coefficient 557 nm) can be linked to the reference peptide by an Ala-Lys linker sequence (Freedman and Radda, 1968). A trypsin digestion, usually performed to produce a peptide mixture from total lysine, here also splits the reference peptide at the COOH terminal linkage of the lysine residue, thereby releasing the dye and linker from the remaining peptide (X).

If protein digestion is performed in H ₂ ¹⁸ O in the presence of a colored reference peptide, the released reference peptide will also be isotopically labeled at its free COOH terminus. . Thus, tryptic digestion of the colored peptide is performed separately in H ₂ ¹⁶ O and is simply added to the whole peptide mixture at the end of the digestion. The linker between the dye and the reference peptide can also be chemically resolved under conditions where the remaining peptides are not affected.

Example 18: Analysis of the proteome of Esherichia coli (strain K12) 10 ⁹ E. coli K12 cells were removed from the cultured stationary growth phase, pelleted by gentle centrifugation, pH 7.2, 20 mM phosphate buffer. Washed 4 times with 1 ml of 10 mM NaCl in solution and lysed by sonication in 1 ml of 4 M urea in 100 mM phosphate buffer at pH 8.0. The lysate was clarified by centrifugation at 100,000 × g in an Airfuge, after which the urea concentration was reduced to 1M using 100 mM phosphate buffer at pH 8.0. To 1 ml of protein mixture (corresponding to 250 · 10 ⁶ E. coli cells) 10 μg trypsin was added and digestion was allowed to proceed overnight at 37 ° C. and stopped by acidification with TFA. One-fifth of the resulting protein-peptide mixture (corresponding to 50 · 10 ⁶ E. coli cells) was equilibrated with 0.1% TFA (solvent A) C18 with an inner diameter of 2.1 mm × 25 cm Loaded on reverse phase HPLC column. The column is first washed for 10 minutes with 5% solvent B (70% acetonitrile in 0.09% TFA) and then from the stationary column phase using a linear gradient of increasing concentration of solvent B. The peptide was eluted. The flow rate was kept at 80 μl / min and a gradient of 1% solvent B / min was set. One minute (ie 80 μl) fractions were collected. The first fraction was collected 18 minutes after the start of the gradient and was numbered 10. This fraction was followed by 39 additional 80 μl fractions, and in the last fraction (fraction 49), the concentration of solvent B reached 63%, corresponding to about 44% acetonitrile. The gradient continued for 37 minutes without fraction collection and ended 105 minutes after the start of the HPLC-run. The UV absorbance profile (at 214 nm) of this run (referred to here as the primary run) is shown in FIG. All collected fractions were vacuum dried and stored at −20 ° C. until further use. The fractions pooled for the secondary run were redissolved in 59 μl of 1% TFA and made up to 0.5% with H ₂ O ₂ by adding 1 μl of 30% H ₂ O ₂ stock solution. The oxidation reaction was allowed to proceed for 30 minutes at 30 ° C., after which the sample was not dried and was used immediately for chromatography. Combine fractions 10, 22, 34, and 46 for the first secondary run (Run 2A) using a single column peptide sorter (see Example 11), and time intervals 4-7, 16-19, 28- Methionine-peptide oxidized within 31 and 40-43 was recovered. For pools of other fractions of the primary run, combinations and recovery times as summarized in Table IVA were used. The UV absorbance profile of a standard secondary run (run 2A with fractions 10, 22, 34 and 46 combined) is shown in FIG. The time interval during which these peptides were collected is shown between the lines in FIG. During this 4 minute period, peptides were collected in 8 consecutive fractions of 30 seconds each (ie 40 μl fraction). In total, 32 fractions of Run 2A were obtained. Eleven consecutive secondary runs were performed to cover the entire peptide set (Table IVA). A suspension of hydrophobic Poros® 50R2 beads was added to the collected fractions (Gevaert et al., 1997) and the fractions were vacuum dried. Peptides concentrated on the added beads were dissolved in 0.7 μl MALDI-matrix solution (4% α-cyanocinnamic acid and 1% −2,5 in 0.1% TFA acetonitrile (1/1). Desorbed in (containing dihydroxybenzoic acid) and transferred to MALDI-target for peptide mass spectrometry in reflectron mode (allowing easy monitoring and confirmation of methionine-sulfoxide containing peptides). FIG. 15 shows MALDI-RETOF-MS spectra of two fractions from secondary run 2A where only methionine-sulfoxide peptide was observed as expected. After analysis of all peptides present in the recovered 320 secondary fractions, we were able to measure 1720 different peptides, including 1618 containing at least one methionine residue. . The measured masses of these 1618 peptides are listed in Table IX. FIG. 18 shows the number of Met-SO-peptides (blue) versus peptides (red) that could not be demonstrated to contain oxidized Met for all fractions of the primary run. This is due to the fact that the standard neutral loss of methanesulfenic acid was not clearly observed or some non-methionine containing peptides slipped during the sorting process. In this embodiment, we have confirmed that 94% of the sorted peptides are composed of peptides that can confirm the presence of Met. This indicates that our sorting system has specificity even when whole cell lysates are used.

In the second step, the sorted peptides and their corresponding parent proteins were identified. Therefore, again, 1 ml of protein mixture prepared from 250 · 10 ⁶ E. coli K12 cells in 1M urea and 0.1M phosphate buffer pH 8.0 was digested. 10 μg trypsin was added and digestion was allowed to proceed overnight at 37 ° C. At the end of the digestion, the resulting protein peptide mixture was reduced with tributylphosphine for 5 minutes and acidified with formic acid (final concentration 1%). A one-fifth of the resulting protein peptide mixture (corresponding to 50 · 10 ⁶ E. coli cells) was applied on a C18 reverse phase HPLC column (ID 2.1 mm × 250 mm; Vydac 218MS52). The column was equilibrated with 0.05% HCOOH as solvent A. The column was first washed with 100% solvent A for 10 minutes. A linear gradient of 1% solvent B / min (solvent B is 0.05% HCOOH in 70% acetonitrile) was used to elute the peptide at a flow rate of 80 μl / min. Peptide UV absorbance profiles were recorded at 214 nm using a UV detector (Applied Biosystems Inc., 759A absorbance detector). A 1 minute fraction was collected. This fraction was collected 30 minutes after the start of the gradient and was numbered 30. A further 50 fractions were collected up to number 80. All collected fractions were vacuum dried and stored at −20 ° C. until further use. The fractions pooled for the secondary run were redissolved in 59 μl of 1% TFA and made up to 0.5% with H ₂ O ₂ by adding 1 μl of 30% H ₂ O ₂ stock solution. The oxidation reaction (alternate process for methionine as a specific amino acid according to the present invention) is allowed to proceed for 30 minutes at 30 ° C., after which the sample is not dried and immediately on the RP-column for use in the secondary run Call. Using the single column peptide sorter described in Example 11, fractions 41, 54, 67, 80 from the primary run were combined, with time intervals 31-39, 44-52, 57-65 and 70--, respectively. Peptides oxidized within 78 (flagged peptides) were recovered. The pooled fractions of the primary run and the fractions collected from the secondary run are listed in Table X. In contrast to our previous example, where flagged peptides were collected at 4 minute time intervals, here at 8 minute intervals (8 fractions of 80 μl each) and 1 minute before elution of unmodified peptide The flagged peptide was recovered. Thereby, W ₁ = 1 minute, δmax = 10 minutes, δmin = 1 minute and W ₂ = 8 minutes. Since each secondary run contained 4 combined windows (W ₂ ), Met-SO peptides (and thus methionine modified flagged peptides) were recovered on 32 fractions of 80 μl each. All odd numbered secondary fractions were then combined, dried and redissolved in 45 μl of solvent A (= 0.05% HCOOH). Half of this mixture was loaded onto a 0.075 mm ID (15 cm long) nanocolumn (C18, Pepmap LC Packings) connected to a trapping column and a 120A HPLC analyzer from Applied Biosystems Inc. A gradient from 0% B to 100% B was formed in 220 minutes using 0.05% HCOOH as solvent A and 0.05% HCOOH in 70% acetonitrile as solvent B. Using a flow splitter (Acurate, LC Packings), the pre-splitter solvent flow rate of 60 μl / min was reduced to about 22 nl / min. The elution peptide was placed in the Z-spray ion source of a Q-TOF mass spectrometer (Micromass UK Limited, Altincham, UK) with a metal coated fused silica needle (FS360-20-10-D-5-C7, New Objective). Introduced. Data were analyzed in data-dependent recovery mode using Masslynx NT (versions 3 and 4). Only double charged ions were automatically selected for MS / MS analysis. The threshold was set at 40 counts / second and selected ions were fragmented by collision with argon atoms. All MS / MS spectra were accumulated and analyzed by MASCOT (Matrix Science Ltd. London) using a protein database containing only E. coli proteins. Obvious identification relied on MASCOT's "Probability Based Mowse Score" (Perkins et al., 1999). The other half of each fraction was resubmitted to nano LC-MS / MS using an exclusion list containing all the double charged ions detected in the previous run. The threshold was now set to 25 counts / second and the selected ions were fragmented for 5 seconds. The same procedure was repeated for even numbered fractions. All protein identification data was finally combined. A total of 6437 CID-spectrums were generated from all nano LC-MS / MS runs (Table XI). These CID spectra resulted in 2543 annotated spectra after submission to the MASCOT server.

  Identification of the sorted Met-SO peptides in all fractions led to the identification of about 767 different E. coli peptides (Table XII). All proteins were covered by an average of 2.2 methionine-containing peptides per protein.

  We are next to a family of secondary proteins such as aminoacyl-t-RNA synthetases, and then a lac-repressor (identified by three independently isolated Met peptides) and at least 19 other Next to very minor proteins such as repressors, all detectable ribosomal proteins that accounted for approximately 10% of the total protein mass of E. coli were identified. These results illustrate the range of dynamic ranges achieved by the present invention that allows detection of low abundance proteins in the presence of the major protein. In addition, we also identified a number of proteins with high hydrophobic profiles and known membrane proteins, suggesting better access to a vast array of biologically important membrane proteins.

When doubled amounts of E. coli cells (100 · 10 ⁶ cells) were analyzed by conventional 2D gel analysis followed by MALDI-based protein identification, 86 proteins were identified. There is at least 10 times and perhaps much higher sensitivity for the latter compared to 767 proteins in gel-free studies. Contamination with human skin keratin, which is often pointed out as a “classical contaminant” when running 2D gels, is greatly reduced when using the method of the present invention and even completely absent. It is important to emphasize that. These analyzes were performed with an equivalent of 50 · 10 ⁶ E. coli bacteria. This corresponds to the amount of protein present in animal cells of ± 50,000 to 100,000 and illustrates the high sensitivity of this technique. The present invention makes it possible to measure the proteome starting from a small number of cells. This makes it possible to analyze differential protein expression in a situation that was not within reach in conventional application fields. The present invention enables analysis of protein expression in small tumor biopsies, in small subregions of the brain, in cells selected by cell sorting, in small subregions of the heart, and in plaque-forming loci in blood vessels To. The method of the present invention efficiently sorts methionine peptides from a highly complex mixture. In addition, flagged peptides are not obtained at once, but are progressively sorted across a number of fractions, thereby feeding the mass spectrometer in a continuous fashion that ensures much more efficient detection. This is best illustrated by the detection of 1618 different Met-peptides from the E. coli proteome using MALDI-TOF-MS detection of the secondary run eluate.

If desired, the method of the present invention can be accomplished without the use of toxic or corrosive chemicals. For example, it is possible to replace acetonitrile with ethanol and TFA with NH ₄ Ac buffer without affecting the overall sorting quality.

Example 19: Partial proteomic analysis of human plasma As starting material, 1 ml of lyophilized human plasma (containing about 60 mg of protein material and essentially free of contaminating cells) was used. The dried sample was redissolved in 1 ml of freshly prepared 8M urea containing 50 mM Tris-HCl pH 8.7 and 2% tributylphosphine. Prior to digestion, the urea concentration was diluted 4-fold by adding 3 ml of 50 mM Tris-HCl buffer (pH 8.7). One fraction of this sample, 200 μl (which corresponds to about 3 mg of protein material) is used for protein digestion with 20 μg trypsin (Sequencing grade modified trypsin from Promega, Madison, WI. USA). did. Digestion was continued overnight at a constant temperature of 37 ° C. and stopped by acidification. By passing the peptide over a Sample Cleanup RP-Column (Agilent Technologies) (ID 2.1 mm × 20 mm, Vydac C ₁₈ RP-bead packed) using a steep gradient of acetonitrile in 0.1% TFA. Half of this digestion mixture was preconditioned. A linear gradient from 0% solvent B (70% acetonitrile in 0.1% TFA in water) to 100% solvent B was generated at a flow rate of 0.2 ml / min for 14 minutes. All eluates were collected, dried under vacuum and redissolved in 100 μl of solvent A (0.1% TFA in water).

  The protein peptide mixture was then subjected to a sorting process. After applying the peptide mixture, 0.1% TFA in water (analyzed by Baker HPLC, Mallinckrodt Baker, 20 minutes at a constant flow rate of 1 ml / min using a Waters ACTION analyzer (Waters Corporate, Milford, MA, USA). The column was washed off using BV, Deventer, Netherlands) (solvent A). Then, using a linear gradient (thus increasing 1% solvent B per minute) to 70% acetonitrile (Baker HPLC analyzed) in 0.1% TFA in water (100% solvent B) over 70 minutes. The peptide was eluted from the RP column. In the final step, the column was flushed with solvent B and re-equilibrated with solvent A prior to the next sample injection. In run 1, the peptide eluting within a time frame between 28 minutes (corresponding to 11.4% solvent B or 8% acetonitrile) and 70 minutes (71.4% solvent B or 50% acetonitrile) Was collected in a 1 minute (ie 1 ml) fraction using a Gilson 221XL Liquid Handler (Gilson SAS, Villers Le Bel, France). Thereby, a total of 42 primary fractions were collected.

Prior to oxidation of the methionine residue, each primary fraction was dried until completely dry. The primary fraction pooled for the secondary run (with a setup similar to that described in Table IVA) was redissolved in 100 μl of 1% TFA plus ₂ μl of 30% H ₂ O ₂ . The oxidation reaction of the methionine residue is allowed to proceed for 30 minutes at 30 ° C., after which the primary fractions are pooled and applied on the same RP-HPLC column as used for the primary separation, under the same chromatographic conditions as in the primary run. Peptides were fractionated in the secondary run. Here, between 9 minutes to 1 minute prior to elution of the unmodified peptide, the flagged peptides were collected at 8 minute time intervals (8 subfractions of 1 ml each). LC-MS / MS analysis was performed on peptides sorted from the two primary fractions. Thus, all the collected sorted peptides from the primary fraction 25 were combined, dried and redissolved in 200 μl solvent A (0.05% formic acid in water), and 1 / 20th of this mixture was added to the trapping column. And an applied biosystems Inc. 120A HPLC analyzer on a nano column (C18 Pepmap, LC Packings) with an inner diameter of 0.075 mm (length 15 cm). The same was done for all sorted peptides from primary fraction 26.

  A gradient from 0% B to 100% B was formed in 220 minutes using 0.05% HCOOH as solvent A and 0.05% HCOOH in 70% acetonitrile as solvent B. The flow splitter (Acurate, LC Packings) was used to reduce the pre-splitter solvent flow rate to 60 μl / min to about 200 μl / min. Introducing eluted peptide into the Z-spray ion source of a Q-TOF mass spectrometer (Micromass UK Limited, Altincham, UK) with a metal-coated fused silica needle (FS360-20-10-D-5-C7, New Objective) did. Data were analyzed in a data dependent collection mode using Masslynx NT (versions 3 and 4) and double charged ions were automatically selected for MS / MS analysis. The threshold was set at 40 counts / second and selected ions were fragmented for 4 seconds by collision with argon atoms.

  All MS / MS spectra were accumulated and analyzed by MASCOT (Matrix Science Ltd, London) using the SWISS-PROT protein database (Release 40, 10) and limiting the search to human proteins. Protein identification relied on MASCOT's "probability-based Mowse score" (Perkins et al., 1999). After the first LC-MS / MS run, an ion exclusion list was created and used for subsequent LC-MS / MS runs to increase the number of peptides analyzed. Here, the threshold was now set to 25 counts / second and the selected ions were fragmented for 5 seconds.

  The resulting protein identification data from these four LC-MS / MS runs are combined and shown in Table XIII. As can be seen, high to moderate abundance plasma proteins such as serum albumin (concentration of about 3-4 g per 100 ml), alpha-microglobulin, apolipoprotein B-100 and fibrinogen beta chain are the splicing factor U2AF35 kDa. Next to unexpected (nuclear) proteins such as subunits and zinc finger proteins. This limited analysis of the human plasma proteome clearly demonstrates the high dynamic range of this technology: a high abundance protein is identified next to a very rare protein. Furthermore, it is important to show that secondary proteins can be identified without prior removal of major components such as plasma albumin and antibodies. Furthermore, the corresponding volume of plasma used for these LC-MS / MS studies is in the range of 1 microliter, which illustrates the ultimate sensitivity of this technology.

Example 20: Partial proteomic analysis of human platelets Contains approximately 500 x 10 ⁹ platelets. A single human blood equivalent of buffy coat cell material was divided into two equal fractions and subjected to centrifugation at 1000 × g for 10 minutes. The pelleted platelets were washed 3 times with 10 ml of TyrodeI buffer from which BSA had been removed, each time followed by a centrifugation step at 1,000 × g for 10 minutes, and finally, TyrodeI without 10 ml of BSA in total. Suspended in buffer (Ardlie et al., 1970). Platelet suspension was added by adding 10 ml of 0.5% Triton X-100 in 25 mM sodium phosphate buffer pH 7.5 containing a cocktail of protease inhibitors (Complete ^™ , Roche Diagnostics GmbH, Manheim, Germany). The turbid solution was dissolved. The lysed platelet suspension is centrifuged at 10,000 × g for 10 minutes to remove cytoskeletal fraction (Fox et al., 1993) and then 2.5 ml of protein mixture (ie 62.5 × 10 6). ⁹ platelet equivalents) in 3.5 ml of 10 mM sodium phosphate buffer at pH 9.0 on a Sephadex® G-25M column (PD-10 column, Pharmacia Bioteck AB, Uppsala, Sweden). Desalted. The desalted protein mixture was concentrated to 1 ml in a centrifugal vacuum concentrator, boiled in a water bath for 5 minutes, and placed on ice for 15 minutes. A protein peptide mixture was generated by digesting the protein overnight at 37 ° C. with 20 μg trypsin (Sequencing grade modified trypsin from Promega, Madison, WI, USA).

On a narrow bore reversed-phase ZOR BAX® 3000SB-C18 column (inner diameter 2.1 × 150 mm, Agilent Technologies Waldbronn, Germany) coupled to an Agilent 1100 Series capillary LC system under the control of the Agilent Chem Station software module A 50 μl fraction of protein digest corresponding to protein material extracted from approximately 3 × 10 ⁹ platelets was injected. After sample injection, the solvent gradient was developed with a steady flow of 80 μl / min. First of all, the column is flushed with 0.1% TFA in water (Baker HPLC analyzed, Mallinckrodt Baker BV, Deventer, The Netherlands) (solvent A) for 10 minutes and then for 100 minutes (hence 1% solvent B / minute). Linear gradient to 70% acetonitrile (Baker HPLC analyzed) in 0.1% TFA (solvent B) was continued (primary run). Starting at 40 minutes (relative to 30% concentration of solvent B), peptides were collected in the form of a total of 48 fractions of 1 minute each (or 80 μl) in a microtiter plate using an Agilent 1100 Series fraction collector. Fractions separated by 12 minutes (see Table XIV) were pooled and dried to complete dryness in a centrifugal vacuum concentrator.

The dried fraction was redissolved in 70 μl of 1% TFA in water and placed in an Agilent 1100 Series Well plate sampler. By transferring 14 μl of freshly made 3% aqueous H ₂ O ₂ solution to a vial containing the peptide mixture, the methionine oxidation reaction proceeded automatically in this compartment. The reaction proceeded for 30 minutes at a constant temperature at 30 ° C., after which the sample was immediately injected onto an RP-HPLC column. Under certain given experimental conditions, methionine-sulfoxide-containing peptides generally elute within a time frame of 7 min to 1 min (see Table XIV) prior to the equivalent time of the corresponding primary fraction, 8 Recovered in the form of two subfractions. After recovery of the Met-SO-peptide, all sub-fractions of the same number are pooled (eg, fractions 12.1, 24.1, 36.1 and 48.1 are pooled for run 2A (Table XIV)). D) Completely dried before LC-MS / MS analysis.

Peptides present in the pooled and dried sub-fraction of a single secondary run were dissolved in 20 μl of 0.1% formic acid (solvent A) in a mixture of acetonitrile / water (2/98 by volume). 10 μl of this was trapped using a CapLC system (Micromass UK Limited, Cheshire, UK) at a flow rate of 20 μl / min of solvent A (total charge time 5 minutes), IDO 3 mm × 5 mm trapping. Automatically injected onto the column (Pep Map, LC Packings, Amsterdam, Netherlands). By switching the stream valve, the tapping column is backflushed with a binary solvent gradient that starts simultaneously with the injection cycle, thereby allowing a nanoscale reverse phase C18 column (ID 0.75 × 150 mm Pepmap ^™ column). , LC Packings). The solvent delivery system was run at a constant flow of 5 μl / min and 200 nl / min of solvent was directed into the nano-column using a 1/25 flow splitter. Peptides were eluted from the stationary phase using a 0-100% solvent B gradient applied at 25 minutes. The outlet of the nanocolumn is a metal-coated fused silica distal PicoTip ^™ needle (PicoTip ^™ FS360-20-10-D-) installed in front of the inlet of Q-TOF mass spectrometry (Micromass UK Lmited, Cheshire, UK). C7, New Objective, Inc., Woburn, MA, USA). Fifteen minutes after switching the stream valve, automatic data dependent recovery in Q-TOF mass spectrometry was started. Recovery parameters were selected in such a way that only double and triple charged ions were selected for fragmentation. The stream valve was switched back 51 minutes after the start of the injection cycle.

  The CID spectra obtained within each LC-MS / MS run were automatically converted to Mascot acceptable format (pkl-format) using Proteinlynx obtained from Micromass Masslynx software (versions 3 and 4). The CID-peak list was used for protein identification in a locally stored database containing only SWISSPROT (Release 40, 10) human sequences using the Mascot algorithm. The following search parameters were used: enzyme; trypsin, maximum number of missing partitions: 2, fixed modification: none, variable modification: oxidation (M), pyro-Glu (N-terminal E and Q), peptide tolerance: 0 .3 Da, MS / MS tolerance: 0.25 Da and peptide charge; 2+ or 3+. To obtain a final list of identified peptides, batch processing of the result set from Mascot was performed. Only the peptides ranked first by Mascot were kept, and those with lower scores than the identity or homology threshold were discarded.

  Table XV presents the results obtained by analyzing methionine-sulfoxide-containing peptides sorted from 8 primary fractions in 2 secondary runs. A total of 16 LC-MS / MS analyzes were performed to sequence the flagged peptides. A MASCOT database search algorithm was used to identify 201 peptides containing at least one Met-SO-residue. Several flagged peptides, especially highly abundant platelet RPs such as actin and myosin, are present in successive subfractions, which represent 98 unique MetSOs at the time of data “cleaning”. Explains the fact that the peptide could be withheld. These MetSO-peptides corresponded to 74 different proteins (see Table XV). Several of the known high-abundance platelet proteins such as myosin, alpha-actin, talin, dyneculin, and actin have been identified by a number of peptides, but most proteins are identified using only one peptide sequence I was able to. It should be emphasized that some of these proteins are large in size (eg talin (molecular weight 270 kDa) and myosin heavy chain (molecular weight 226 kDa), which are rarely detected on 2-D gels. is important.

  The dynamic range of our sorting technique for proteome analysis is already evident in this limited data set. For example, low-abundance proteins that are difficult to detect on 2-D gels, such as ras-related proteins, are probably actin, tubulin, tropomyosin, talin and at least 1000-fold higher in these cells. It is identified next to highly abundant proteins such as myosin. Importantly, five different isoforms of these proteins (RAC1-HUMAN, RALA-HUMAN, RAPB-HUMAN, RB5A-HUMAN and RB5B-HUMAN) have been identified (Table XV), one of which is RB5A-HUMAN Was identified even with two differently flagged peptides.

  One class of proteins that are difficult to detect on 2-D gels are hydrophobic proteins. Among our restricted platelet proteomes, we have found that LIM and SH3 domain protein 1 (GRAVY value -1.02), carmenin precursor (GRAVY), which is usually hardly detected on 2-D gels due to its hydrophobicity Proteins with very high hydrophobicity, such as value -1.01) and moesin (GRAVY value -0.98) were identified.

Example 21: Human platelet proteome acetylated amino-terminal arginine ending peptide As starting material, cytosolic and membrane scaffold preparations as prepared in example 20 were used. In a centrifugal vacuum concentrator, 1.5 ml of the desalted protein mixture (about 9 mg or an estimated amount of 300 nmol total protein material) was dried to about 1 ml. To this protein mixture, solid guanidinium hydrochloride was added to a final concentration of 4M. Protein was reduced by adding 40 μl of 0.5% tributylphosphine in n-propanol to this mixture and incubating at ambient temperature for 30 minutes. 188 μl of freshly prepared 40 nmol / μl iodoacetamide solution was added to the reduced protein mixture and the protein was alkylated for 90 minutes at 37 ° C. in the dark. The protein solution is diluted with water to a total volume of 1.5 ml, after which 500 μl of this mixture is desalted on a NAP ^™ -5 column (Amersham Pharmacia Biotech) and 1 ml of 250 mM Tris.HCl, pH 7.9, containing 250 mM guanidinium hydrochloride. Was recovered. The desalted protein mixture is concentrated to half its volume by vacuum drying, boiled in a water bath for 5 minutes, placed on ice for 10 minutes, then 10 μg trypsin (sequencing grade modified trypsin from Promega) Was added. Proteolytic digestion was allowed to proceed overnight at a constant temperature of 37 ° C. and stopped by acidification with TFA.

  After centrifugation to remove any insoluble material, the resulting peptide mixture was separated on a reverse phase HPLC column (4.6 ID x 250 mm RP-HPLC C18 column, Vydac Separations Group). After injecting the sample onto the column, the peptide mixture was fractionated using an increasing concentration gradient of acetonitrile. First, the column was washed with 0.1% TFA in water (Baker HPLC analysis) (solvent A) for 5 minutes with a constant flow of 1 ml / min using a Waters Gradient Controller and a Waters Model 510 solvent pump. Then, using a linear gradient to 70% acetonitrile (Baker HPLC analyzed) in 0.1% TFA in water (100% solvent B) over 70 minutes (and thus 1% acetonitrile increase per minute), the RP column The peptide was eluted from. In the final step, the column was thoroughly rinsed with solvent B and re-equilibrated with solvent A prior to the next sample injection. The peptide eluting between 2 minutes (corresponding to 0% solvent B) and 66 minutes (corresponding to 87.1% solvent B and 61% acetonitrile) is formed into 16 ml of 16 primary fractions of 4 ml each. It was collected at.

  All primary fractions were dried in a centrifugal vacuum concentrator until completely dry and redissolved in 1 ml of 50 mM sodium borate, pH 9.0. For each primary fraction, half of the peptide mixture is used to block the peptide at its free amino group by adding 3 μl of 0.1 M aqueous 2,4,6-trinitrobenzenesulfonic acid (TNBS) (Sigma). While the other half was used as a control. The reaction was allowed to proceed for 60 minutes at 37 ° C., after which an additional 3 μl of 0.1 M TNBS was added and incubated again at 37 ° C. for 60 minutes. Most of the TNB-peptide as well as the reaction byproducts of TNBS (eg, picrate) were extracted with 500 μl of ethyl acetate (equilibrated with water). This extraction procedure was repeated twice. The aqueous phase containing the arginine ending peptide without N-terminal blocked (acetylated) lysine was dried under vacuum. As described above, the TNBS-modification reaction is repeated a second time to allow the remaining small amount of peptide with a free amino group to react. The dried product is dissolved in solvent A and the peptides within a total window of 7.5 minutes (starting 2 minutes before the start of recovery of the original primary fraction) in the form of 500 μl each of a total of 15 subfractions. Is attached to the recovered secondary run. Each sub-fraction was dried and redissolved in 20 μl of 0.1% formic acid in a mixture of acetonitrile in water (2/98 by volume) (of which 10 μl was used for LC-MS / MS analysis) Used for protein identification as described in Examples 19 and 20.

  For MASCOT-based database searches, the following search parameters were used: enzyme: trypsin, maximum number of missing divisions: 2 fixed modification: none, variable modification: acetylation (N-terminal), oxidation (M), pyro- Glu (N-terminal E and Q), peptide tolerance: 0.3 Da, MS / MS tolerance: 0.25 Da and peptide charge: 2+ or 3+. To obtain a final list of identified peptides, batch processing of the result set from Mascot was performed. Only the peptides ranked first by MASCOT and meeting their identity and / or homology threshold are retained and combined in Table XVI with their corresponding precursor peptides. As expected, the naturally blocked (acetylated) N-terminal peptide ending with an arginine residue is followed by peptides starting with pyroglutamic acid and starting with a proline residue. The former results from the formation of a cyclic blocking residue, while the N-terminal proline forms a secondary amine that does not react with TNBS.

  In addition to the identified peptides, we present a list of 183 newly derived peptide sequence tags from the MS / MS-spectrum that did not lead to obvious identification in the SWISSPROT database using the MASCOT database search algorithm. (Table XVII). Most of the derived tags are homology-based search tools such as BLAST and FASTA. They are most likely to represent the acetylated N-terminal peptide of the protein such that the corresponding sequence is data not listed in the available sequence database.

Example 22: In contrast to the limited proteome analysis related example is based on the isolation of NH ₂ -terminal peptides of the proteins present in human platelet extracts, we here, those having an amino-terminal blocked and The NH ₂ -terminal identification peptides of proteins present in the sample, including those with a free amino terminus, were isolated and modified in alternation chemistry so that they could be used for protein identification.

As starting material we used cytosolic and membrane scaffold preparations of human platelets as prepared in Examples 20 and 21. In a centrifugal vacuum concentrator, 500 μl of the desalted protein mixture (estimated amount about 3 mg) was concentrated to about 400 μl. To this protein mixture, guanidinium hydrochloride was added to a final concentration of 4M. The protein was reduced by adding tributylphosphine (14 μl of a fresh 0.5% solution in n-propanol) for 30 minutes at ambient temperature. 62.5 μl of a 40 nmol / μl iodoacetamide solution in freshly prepared H ₂ O was added to the reduced protein mixture and the protein was alkylated for 90 minutes at 37 ° C. in the dark. The mixture was then desalted on a NAP ^™ -5 column (Amersham Pharmacia Biotech) and recovered in 1 ml of 250 mM sodium phosphate buffered at pH 8.0 containing 1 M guanidine hydrochloride. This volume was concentrated in half in a centrifugal vacuum concentrator. Both α- and ε-amines were acetylated by adding a 50-fold molar excess of solid sulfol-hydroxysuccinimide acetate and incubating the mixture for 90 minutes at room temperature. The possible acetylation of hydroxyl and COOH groups was reversed by adding 1 μl of hydroxylamine to the protein reaction mixture. Prior to proteolysis, the protein mixture was desalted onto a NAP ^™ -5 column and recovered in the form of a total of 1 ml of 50 mM Tris.HCl, pH 7.9, containing 250 mM guanidine hydrochloride. The desalted protein mixture is concentrated by vacuum drying to half its volume, boiled in a water bath for 5 minutes, placed on ice for 10 minutes, and then 10 μg trypsin (sequencing grade modified from Promega). Added trypsin). Proteolytic digestion was continued overnight at a constant temperature of 37 ° C. and stopped by acidification with TFA.

  The sorting process for the acetylated amino terminal peptide was carried out on the same RP-HPLC column under the same conditions as described in Example 21. Shown in Table XVIII are the results obtained after LC-MS / MS analysis of amino-terminal peptides sorted from the two primary fractions (9 and 10). This accounts for 1/8 of the total number of fractions. Again, the MASCOT algorithm was used to identify fragmented peptides and the parameters are now described in Example 21, except that acetylation of lysine residues was an additional variable modification. Set in the same way as

  This partial proteomic analysis (only 12.5% of all analyzable material was used) yielded 26 distinct proteins that could be identified (see Table XVIII). Interestingly, major proteins such as actin are identified next to hydrophobic proteins such as DAD-1 protein, which are predicted to be of low abundance such as kinases or phosphatases and essential membrane proteins. The Furthermore, as in Example 21, we analyzed a certain number of peptide ions that did not lead to any identification using the MASCOT algorithm but gave an interpretable fragmentation spectrum. These spectra were newly interpreted, but the resulting 48 peptide sequence tags (shown in Table XIX) were revealed using sequence homology-based database search tools such as FASTA and BLAST. Did not lead to any identification. We assume that these sequences represent novel proteins with sequences that are not yet available in the database.

Example 23: Specific isolation of COOH-terminal peptides of proteins in complex mixtures The procedure begins with the conversion of protein cysteines with iodoacetamide or similar SH-specific reagents known in the art. The protein mixture is then digested with trypsin to produce a protein peptide mixture. Thereafter, all the COOH groups and esters, including the COOH terminus of the peptide derived from the COOH terminal portion of the protein and the Arg and Lys COOH groups at the end of the peptide, and diazo derivatives forming tyrosine and ether, Process the entire peptide mixture.

  These pre-treated peptides are then separated by conventional or reverse phase chromatography and the eluted peptides are modified peptides from unmodified peptides in the secondary run in each of the collected fractions. Are recovered in the form of a number of fractions so as to allow separation. While trypsin is added in each fraction to reverse hydrolyze the ester at the Arg and Lys residues, other COOH esters, including esters at the COOH terminus of proteins that are generally composed of Arg or Lys residues, It is not reverse hydrolyzed by trypsin. Thereby all the tryptic peptides except the COOH terminal peptide are modified and shifted into the secondary run. COOH-terminal peptides are thereby recovered in the secondary run as unmodified peptides within the same time interval as they eluted during the primary run.

  Candidate diazo derivatives can be highly reactive and toxic diazomethane or phenyldiazomethane or more ideally non-volatile and more stable water-soluble diazo derivatives. All of these compounds react to the COOH group and its corresponding ester, and to the phenol group and the corresponding ether.

  Esters of Arg or Lys COOH groups are trypsin substrates that are hydrolyzed in a manner similar to the corresponding peptide bond.

  The hydrolysis reaction of the COOH-terminal Lys benzoyl ester is depicted in schema (xi).

Abbreviations 2D: Two-dimensional CAD: Collision-activated dissociation DTT: Dithiothreitol EST: Electrospray ionization EST: Expressed sequence tag FTMS: Fourier transform mass spectrometry ICAT: Isotope-coded affinity tag ID-peptide: Identification Peptide IPG: immobilized pH gradient ITC: isothiocyanate LC: liquid chromatography LC-MS / MS: liquid chromatography and tandem MS
MALDI-RETOF-MS: MALDI-Reflectron TOF-MS
MALDI-TOF-MS: Matrix Assisted Laser Desorption / Ionization-Time of Flight-Mass Spectrometry Met-SO: Methionine-Sulphoxide MS / MS: Tandem MS
MS: Mass Spectrometry MW: Molecular Weight PI: Isoelectric Point PITC: Phenyl Isothiocyanate PMF: Peptide Mass Fanger Print PSD: Post Source Decomposition PTC: Phenylthiocarbamyl RP-HPLC: Reversed Phase High Performance Liquid Chromatography SDS: Sodium Dodecyl Sulfate TFA: trifluoroacetic acid

Table I Predicted protein percentage lacking trace amino acids. All species protein sequences were extracted from SwissProt, Version 39. Met (-onset) means the number obtained after the starting methionine has been removed.

  Table II: The principle of the peptide sorter has been devised by way of example for flagged peptides. The fraction is isolated in run 1 with an elution window W1 equal to X / 2. If the total run 1 elution window of all peptides derived from the protein peptide mixture is equal to 20X, then 40 fractions with X / 2 windows (first fraction: 0 to X / 2; second fraction: X / 2 to X; third fraction: X to 3X / 2 ...) is recovered. If a specific amino acid modification in the flagged peptide induces a forward shift on the hydrophobic separation column and the value of this shift varies between X and 2X, this is the value for δmin = X / 2, δmax = 5X / 2, W1 = X / 2 and W3 = 7X / 2). The general principle of the parallel sorter can be illustrated by an example of generating 12 peptide fraction pools. Thereby, after the primary chromatography run, the following fractions can be pooled: Fraction 1 (0-X / 2) and Fraction 13 (6X-13X / 2), 25 (12X-25X / 2) and 37 ( 18X-37X / 2). Similarly, fraction 2 is pooled with fractions 14, 26 and 28; fraction 3 is pooled with fractions 15, 27 and 39; fraction 4 is pooled with fractions 16, 28 and 40; fraction 5 is fractions 17 and Fraction 6 is pooled with fractions 18 and 30; fraction 7 is pooled with fractions 19 and 31; fraction 8 is pooled with fractions 20 and 32; fraction 9 is pooled with fractions 21 and 33; 10 is pooled with fractions 22 and 34; fraction 11 is pooled with fractions 23 and 35; fraction 12 is pooled with fractions 24 and 36. The 12 pools are then chemically and / or enzymatically modified on at least one selected amino acid. Table II contains the theoretical shift calculations for the 12 flagged peptide pools.

Table III: hydrophilic shifts of peptide NH ₂ -YSFVMTAEK-COOH due to oxidation to the sulfoxide embodiment. Elution was performed with different organic solvents using a linear gradient of 1% per minute. The buffer composition was either trifluoroacetic acid, ammonium acetate pH 5.7 or formic acid. The column was a C18 reverse phase column (inside diameter 4.6 × 2500 mm) each time. The retention time of the oxidized peptide embodiment is expressed in minutes (RtMET-OX _(min) ) and the shift is expressed in minutes between the non-oxidized peptide and the sulfoxide peptide. (Rt _(min) ). The absolute elution time (RtMET-OX _(min) ) is highly dependent on the nature of the ions used in the system, and the range of the shift (Rt _(min) ) is largely determined by the nature of the organic modifier. The strongest shift was observed when methanol was used as the organic modifier. This was not used in practice because there was a significant peak broadening effect that was not observed for ethanol or acetonitrile as a modification.

  Table IVA: Mode of combining fractions derived from primary runs and collecting fractions between secondary runs. The run number (2A-2L) means the number given to the secondary run. For convenience, the first collected fraction is called n ° 10 (see text). This fraction elutes during the first run between 18 and 19 minutes after the start of the acetonitrile gradient. Consecutive fractions are numbered up to 49, representing fractions of 1 minute.

  Table IVB: Combination mode of fractions derived from secondary runs for separation in tertiary runs. 3A-3D means the number given to the tertiary run. Color refers to that used in Table IVA.

  Table IVC: Combination mode of fractions derived from secondary runs for separation within tertiary runs. Here, an alternative fraction combination method when formic acid is used as the buffer system is shown (3'A-3'H). The color code used in the fraction number is the same as that used in Tables IVA and IVB.

  Table V: Combination mode of fractions derived from the primary run for separation within the secondary run using a three column peptide sorter (Figure 12). 2A-2D means a secondary run and the eluting fraction is either online connected to ESI-based mass spectrometry or can be recovered for further analysis by MALDI-TOF-MS.

  Table VII: Combination mode of fractions derived from the primary run for separation within the secondary run using a 9 column peptide sorter (Figure 13). 2A-2E means a secondary run and the eluting fraction is either online connected to ESI-based mass spectrometry or can be recovered for further analysis by MALDI-TOF-MS.

Valve operation during a complete run of a three column peptide sorter First fractions 10, 22, 34 and 46 are applied as a mixture on column I, fractions 14, 26, 38 and 50 are applied on column II, fractions 18, 30 and 42 are applied on column III. Each input is expected to take a minute. The three columns are then washed simultaneously with 2% solvent B for 10 minutes. A linear gradient then started from 2% solvent B to 55% solvent B, which is 70% acetonitrile in water with 0.1% TFA added. The slope is the same as if it were used in a primary run. Valve open (+); valve closed (-). The valves are numbered ai (high pressure valve) and jo (low pressure dead volume valve) and are shown in FIG. All runs including charging, cleaning ...

Example of valve setting in a 9-column type peptide sorter The operation of valve setting in the system A is described. +: Valve open;-: Valve closed. Fraction 12 of the primary run is applied to column 1, fraction 24 is applied to column II, and fraction 36 is applied to column III. A linear gradient is formed that increases by 1% solvent B per minute. This gradient increases to 55% solvent B. The modified peptide was recovered between 6 and 9 minutes (6-9), 18-21 and 30-33 and is represented by solid bars. A valve is shown. a-g are high-pressure valves, and ho and pr are dead volume low-pressure valves. Lines represent connecting tubing.

  Table X: Combination mode of fractions derived from secondary runs for separation in tertiary runs combined with automated MS / MS analysis. Results from Run 3L are further described in Tables 11 and 12 and in the text.

  Table XI: Table showing the number of MS / MS-spectrums obtained leading to the identification of E. coli proteins

  Table XII: List of proteins identified by LC-MS / MS analysis for MetSO sorted peptides. Proteins are sorted according to their SwissProt input name.

  Table XIII: Complex protein peptide mixtures were generated by trypsin digestion on unfractionated whole human plasma. Met peptides were sorted by oxidation using an equivalent of 1 microliter of plasma. Only two of the ... fractions of the primary run were further analyzed. The identified peptides and corresponding proteins in these fractions are listed. (DB input: available at http: // wwwexpasy. Ch / sprot).

  Which primary fraction for sorting of methionine-containing peptides during the secondary run (second column) under certain given chromatographic conditions (the depicted recovery time is given in column 3) An overview that shows if you can pool. The elution interval for recovery of MetSO-peptide is given in the last column. The Met-SO peptide obtained in Runs 2A and 2F was further used for protein identification (see Table XV).

  Table XV: List of RPs and peptides identified by analyzing Met-SO peptides sorted in 2 secondary runs from a total of 8 primary fractions. The results presented in this table were obtained after 16 LC-MS / MS runs for sorted Met-SO peptides. The protein was obtained from the Triton soluble fraction of human platelets.

  Table XVI: Proteins identified after selection of their acetylated lysine-free N-terminal arginine ending peptides from trypsin digestion of human platelet cytosol and membrane skeletal extracts. The identified peptides are divided into three parts: (1) naturally N-terminally blocked peptides, (2) peptides beginning with proline (also covering the N-terminus of the protein) and (3) (proteins An internal peptide starting with pyroglutamic acid, which is a byproduct of the preparation of the peptide mixture.

  Table XVII: MS / MS from sorted acetylated arginine-containing lysine amino-terminal peptides (Example 21) that did not lead to unambiguous identification of proteins using MS / MS-based database search tools such as MASCOT Newly derived peptide sequence tag from the spectrum. The primary fraction from which the peptides are sorted is shown the peptide mass and the sequence tag derived from N-> C (m = methionine sulfoxide, x = unassigned amino acid).

  Table XVIIIA: Proteins identified after selection of their N-terminal peptides. Two primary fractions were analyzed in trypsin digests of human platelet cytosol and membrane skeletal extracts. A. Identified proteins; List of sequences derived from MS / MS spectra that could not be identified in the available database.

  Table XIX: Newly derived peptide sequences from MS / MS spectra from sorted amino terminal peptides (Example 22) that did not lead to unambiguous identification using a search tool such as MASCOT (k = acetylated) Lysine, x = unassigned amino acid).

FIG. 4 is a chart demonstrating the direct peptide sorting process and showing the different parameters used to describe the sorting process. (A) Total protein peptide mixture separated in the primary run; t3 and t4 represent the time interval considered for a given fraction (w1). (B) The flagged peptide displays a hydrophilic shift between δmin and δmax. They elute between time intervals t1 and t2 within window w2. (C) The flagged peptide is more hydrophobic and exhibits a hydrophobic shift between δ ′ and δ′max and elutes between time t5 and t6 in window w2 ′. Four fractions from the primary run (FIG. 1A) were pooled and subjected to the modification process. These are attached to the secondary run and the flagged peptides are between t1 and t2, between t′1 and t′2, between t ″ 1 and t ″ 2, and t ″ ′ 1 and t ″ ′ 2 is eluted. Fractions are combined in such a way that the sorted peptides do not overlap with unmodified peptides from previous fractions. By pooling fractions, the number of secondary runs is reduced. NH ₂ —YSFV M TAER-COOH (A), NH ₂ before (lower trace) and after (upper trace) treatment with 0.5% H ₂ O ₂ in 1% TFA for 30 minutes at 30 ° C. -YSFV C TAER-COOH (B) and NH ₂ -YSFV W TAER-COOH UV absorbance profile of 214nm of RP-HPLC separation of (C). The MALDI-RETOF-MS spectra of the eluting control and H ₂ O ₂ treated peptides (lower trace vs. upper trace, respectively) are shown in Figures DF. NH ₂ —YSFV M TAER-COOH (A), NH ₂ before (lower trace) and after (upper trace) treatment with 0.5% H ₂ O ₂ in 1% TFA for 30 minutes at 30 ° C. -YSFV C TAER-COOH (B) and NH ₂ -YSFV W TAER-COOH UV absorbance profile of 214nm of RP-HPLC separation of (C). The MALDI-RETOF-MS spectra of the eluting control and H ₂ O ₂ treated peptides (lower trace vs. upper trace, respectively) are shown in Figures DF. NH ₂ —YSFV M TAER-COOH (A), NH ₂ before (lower trace) and after (upper trace) treatment with 0.5% H ₂ O ₂ in 1% TFA for 30 minutes at 30 ° C. -YSFV C TAER-COOH (B) and NH ₂ -YSFV W TAER-COOH UV absorbance profile of 214nm of RP-HPLC separation of (C). The MALDI-RETOF-MS spectra of the eluting control and H ₂ O ₂ treated peptides (lower trace vs. upper trace, respectively) are shown in Figures DF. NH ₂ —YSFV M TAER-COOH (A), NH ₂ before (lower trace) and after (upper trace) treatment with 0.5% H ₂ O ₂ in 1% TFA for 30 minutes at 30 ° C. -YSFV C TAER-COOH (B) and NH ₂ -YSFV W TAER-COOH UV absorbance profile of 214nm of RP-HPLC separation of (C). The MALDI-RETOF-MS spectra of the eluting control and H ₂ O ₂ treated peptides (lower trace vs. upper trace, respectively) are shown in Figures DF. NH ₂ —YSFV M TAER-COOH (A), NH ₂ before (lower trace) and after (upper trace) treatment with 0.5% H ₂ O ₂ in 1% TFA for 30 minutes at 30 ° C. -YSFV C TAER-COOH (B) and NH ₂ -YSFV W TAER-COOH UV absorbance profile of 214nm of RP-HPLC separation of (C). The MALDI-RETOF-MS spectra of the eluting control and H ₂ O ₂ treated peptides (lower trace vs. upper trace, respectively) are shown in Figures DF. NH ₂ —YSFV M TAER-COOH (A), NH ₂ before (lower trace) and after (upper trace) treatment with 0.5% H ₂ O ₂ in 1% TFA for 30 minutes at 30 ° C. -YSFV C TAER-COOH (B) and NH ₂ -YSFV W TAER-COOH UV absorbance profile of 214nm of RP-HPLC separation of (C). The MALDI-RETOF-MS spectra of the eluting control and H ₂ O ₂ treated peptides (lower trace vs. upper trace, respectively) are shown in Figures DF. (A) UV absorbance profile of 214nm reversed phase C18HPLC- separated on column peptide NH ₂ -YSFVCTAER-COOH. The peptide is modified with acrylamide and subsequently oxidized to its S-propionamide-cysteine sulfoxide derivative (bottom trace), which, when run under the same HPLC conditions, is unmodified peptide (top race). ) About 2 minutes of hydrophilicity shift. Note the presence of closely migrating enantiomeric doublets typical of sulfoxide derivatives within the TFA-acetonitrile system. (B) MALDI-RETOF-MS spectrum of the S-propionamide cysteine sulfoxide derivative of the peptide NH ₂ -YSFVCTAER-COOH. Fragment ions arising from the rapid neutral loss of the modified side chain of cysteine residues are shown in blue, which greatly aids in identifying the presence of the modified peptide within the parent peptide. (A) UV absorbance profile of 214nm reversed phase C18HPLC- separated on column peptide NH ₂ -YSFVCTAER-COOH. The peptide is modified with acrylamide and subsequently oxidized to its S-propionamide-cysteine sulfoxide derivative (bottom trace), which, when run under the same HPLC conditions, is unmodified peptide (top race). ) About 2 minutes of hydrophilicity shift. Note the presence of closely migrating enantiomeric doublets typical of sulfoxide derivatives within the TFA-acetonitrile system. (B) MALDI-RETOF-MS spectrum of the S-propionamide cysteine sulfoxide derivative of the peptide NH ₂ -YSFVCTAER-COOH. Fragment ions arising from the rapid neutral loss of the modified side chain of cysteine residues are shown in blue, which greatly aids in identifying the presence of the modified peptide within the parent peptide. An overview of the reaction sequences used for sorting methionine, cysteine and the sum of cysteine and methionine containing peptides. NH ₂ - general description of the major steps in order to sort the subset of endblocked peptide. Critical amino acid residues are represented. R = Arg, K = Lys, hR = homoArg, PIC = phenyl isocyanate, PC = phenylalbamyl. All PTC-peptides become more hydrophobic. The N-acetylated peptide remains unchanged and will elute in run 2 just as it did in run 1. The blocked peptide will thereby be separated from most PTC-peptides. A pie chart showing the number of “unique” peptide masses generated by in-house endoproteinase Lys-C digestion against curated SwissProt protein input from both human and E. coli. As seen in both cases, more than 90% peptide mass (calculated with an accuracy of 0.001 Da) corresponds to a unique peptide sequence containing at least one methionine residue in the database, thereby Can be used to identify their parent proteins. Schematic summary of the reactions leading to the terminal peptides pace proteomic approach - quantitative differential NH _2. Critical amino acid side chains are indicated. R = Arg, K = Lys, hR = homoArg, PIC = phenyl isocyanate, PC = phenylcarbamyl, TNBS = trinitrobenzenesulfonate. ¹⁶ O / ¹⁸ / O means differential labeling obtained by digestion in H ₂ ¹⁶ O or H ₂ ¹⁸ O, respectively. Single column peptide sorter: Peptide sorting occurs during the secondary run after modifications have been made to the fraction of the primary run. Peptide fractions from the primary run were combined as specified in Table IVA and input by the sample injector. All conditions (RP-adsorbent, flow rate, gradient, solvent, etc.) were kept the same in the primary and secondary runs. In the single column version, all peptides pass through the same column. After sample loading, a gradient is created using a conventional commercially available high pressure HPLC pump system (referred to herein as a solvent pump). The valves are automatically steered (a and b are high pressure valves and c and d are low pressure valves), and the solvent flow is analyzed by an analytical instrument (fraction collector, mass spectrometer or MALDI-target) or waste Lead in any desired direction. 50.106 214 nm UV absorbance profile of RP-HPLC separation of total tryptic digest of E. coli cell lysate (C18-column with ID 2.1 × 250 mm). Starting with 5% B, tryptic peptides are eluted by utilizing an increasing linear gradient of 1% B / min at a constant flow of 80 μl / min. Peptides eluting between 23% solvent B and 63% solvent B are collected in 40 μl of 40 fractions each. The first collected fraction is numbered 10 and the last is numbered 49 (see also Example 18). The fraction taken and further processed in FIG. 11 is indicated by a blue open rectangular box. Of the methionine-sulfoxide peptide obtained after mild oxidation of the peptides present in fractions 10, 22, 34 and 46 (primary run) (using 0.5% H ₂ O ₂ in 1% TFA) 214 nm UV absorbance profile showing recovery. Met-SO peptide recovery begins 6 minutes before elution of large amounts of unmodified peptide and lasts for 4 minutes. Chromatographic conditions were the same as shown in FIG. Fractions containing unmodified peptides are indicated by blue arrows and sorted peptides are limited in red: 4-7, 16-19, 28-31 and 40-43 (Table IVA). Sorter to triple column: This system works with three identical RP columns connected in parallel. Again, all conditions remain the same, not only during parallel runs, but also compared to primary runs. The fractions recovered from the primary run were combined, modified and distributed on each column as specified in Table V. The solvent stream is kept constant throughout the three columns. This connects each individual column to a high pressure pump system, thereby using three such pumps (version A) or using a single high pressure pump to control a controlled splitter system. And can be achieved by controlling the flow rate towards each column (version B). Such flow regulators are currently commercially available (valve ai is a high pressure valve and valve jo is a low pressure valve). These valves can be steered by a PC and allow fully automated operation including loading, separation and analysis (fraction collection, mass spectrometer or MALDI-target). Sorter to triple column: This system works with three identical RP columns connected in parallel. Again, all conditions remain the same, not only during parallel runs, but also compared to primary runs. The fractions recovered from the primary run were combined, modified and distributed on each column as specified in Table V. The solvent stream is kept constant throughout the three columns. This connects each individual column to a high pressure pump system, thereby using three such pumps (version A) or using a single high pressure pump to control a controlled splitter system. And can be achieved by controlling the flow rate towards each column (version B). Such flow regulators are currently commercially available (valve ai is a high pressure valve and valve jo is a low pressure valve). These valves can be steered by a PC and allow fully automated operation including loading, separation and analysis (fraction collection, mass spectrometer or MALDI-target). Nine-column peptide sorter: This system differs from previous devices in several ways: i) One fraction of the primary run is put on one column each time, ii) Each column is Smaller than the primary run column and may be composed of disposable material, and iii) in series / parallel in such a way that all columns are fully deployed before the gradient is directed towards the next column Operated in combined mode. Since the column is smaller, the operating time can be reduced. Valves ag controlling the column inlet are high pressure valves. Valves ho and pr control different column outlet streams to either waste or analytical systems and may be low pressure dead volume valves. Columns I, II and III are developed with the same solvent gradient, the first part of the gradient is directed to column I, the second part is used for column II, and the third part is column III. (See Example 13 for details). Separation of flagged and unmodified peptides is steered by valve settings that can be manipulated using a PC. A 9 column sorter operated with 3 sets of 3 columns each running with a delay compared to the previous set, and in the example described here, this delay was set to 3 minutes: Started 3 minutes behind A and C started 3 minutes after the start of B. The eluted peptides from each of the column sets are directed toward the analytical tool as described above by valve pr. : (A) peptide reference mixture _{NH 2 -Alan-Arg-COOH (} n = 7~42) UV absorbance profile of RP-HPLC separation of (214 nm). The components of this mixture that differ by an additional 1 alanine residue can be clearly recognized. The separation was performed on a 2.1 mm ID RP-HPLC C18-column using a linear acetonitrile gradient in 0.1% TFA. (B) MALDI-RETOF-MS spectrum revealing the different components present in this mixture separated at 71 amu's. : (A) peptide reference mixture _{NH 2 -Alan-Arg-COOH (} n = 7~42) UV absorbance profile of RP-HPLC separation of (214 nm). The components of this mixture that differ by an additional 1 alanine residue can be clearly recognized. The separation was performed on a 2.1 mm ID RP-HPLC C18-column using a linear acetonitrile gradient in 0.1% TFA. (B) MALDI-RETOF-MS spectrum revealing the different components present in this mixture separated at 71 amu's. MALDI-RETOF mass spectrum of peptides present in two fractions of recovered Met-SO peptide (Figures A and B). Given the mass of the identified Met-SO peptides, their characteristic fragmentation products with the loss of methanesulfenic acid observed in reflectron mode are represented, giving shorter fragments ( Blue arrows) MALDI-RETOF mass spectrum of peptides present in two fractions of recovered Met-SO peptide (Figures A and B). Given the mass of the identified Met-SO peptides, their characteristic fragmentation products with the loss of methanesulfenic acid observed in reflectron mode are represented, giving shorter fragments ( Blue arrows) Diagram of quantitative differential proteomic approach sorting for methionine peptides. MSO means methionine sulfoxide. Diagram of quantitative differential proteomic approach sorting for methionine peptides. MSO means methionine sulfoxide. MALDI-RETOF-MS of 19 peptides (peptide mass on the X axis) obtained from a total of 5 pmol of a 1/1 mixture of tryptic digests labeled with ¹⁶ O- and ¹⁸ O- of BSA. Isotope ratio (X-axis value). For the measured quota of BSA-mix, an average value of 1.03 was obtained. MALDI-RETOF in the primary fractions of a tryptic digest of the protein material from 50, 16 of the ^six E. coli cells in relation to the peptide to contain a methionine could not be demonstrated (shown in red) -Graph depicting the number of Met-SO peptides using MS (shown in blue).

Claims (70)

In a method for isolating a subset of peptides from a protein peptide mixture,
(A) separating the protein peptide mixture into peptide fractions by chromatography; (b) at least one amino acid of at least one of the peptides in each fraction chemically, enzymatically, or chemical and are enzymatically modified, step for generating a subset of peptides made a modified flag, and (c) or if it were the modified from each fraction by chromatography to isolate the peptide made a flag A process comprising the steps, wherein the chromatography of steps (a) and (c) is carried out with the same type of chromatography. The method of claim 1, wherein the chromatographic conditions of steps (a) and (c) are the same or substantially similar. The method according to claim 1 or 2, wherein there is one or more pretreatment steps prior to step (a). The amino acid to be modified is selected from the group consisting of arginine, proline, cysteine, methionine, tryptophan, lysine, tyrosine, histidine, phenylalanine and any combination of two or three thereof. The method described in 1. The method according to claim 4, wherein the amino acid to be modified is methionine. The method according to claim 4, wherein the amino acid to be modified is cysteine. The method according to claim 4, wherein the amino acids to be modified are methionine and cysteine. The method according to claim 1, 2, or 3, wherein the amino acid to be modified is an amino acid modified simultaneously with or after translation. Translational and post-translational modifications are selected from the group consisting of glycosylation, phosphorylation, acetylation, sulfation, ubiquitination, alkylation, nitrosylation, oxidation, hydroxylation, and methylation and any combination thereof 9. The method of claim 8, wherein: The method according to claim 9, wherein the amino acid modified simultaneously with or after translation is a phosphorylated amino acid. 10. The method of claim 9, wherein the amino acid modified simultaneously with or after translation is a glycosylated amino acid. The method according to claim 9, wherein the amino acid modified simultaneously with or after translation is an ε-N-acetylated amino acid. 13. The method of claims 1-12, further comprising identifying a flagged peptide and its corresponding protein. 14. The method of claim 13, wherein the identifying step is performed by a tandem mass spectrometer in combination with a database search. The method of claim 13, wherein the identifying step is performed by post-source decomposition analysis combined with database search. 14. The method of claim 13, wherein the identifying step is performed by measuring peptide mass in combination with a database search. The identification step further comprises (a) the presence of a modified amino acid; (b) the determination of the number of free amino groups in the flagged peptide; (c) the resolution of the protease used to produce the protein peptide mixture. 17. The method of claim 16, wherein the method is based on knowledge of specificity; and (d) one or more of the large averages of peptide hydrophobicity. In a method for determining the relative amount of at least one protein in more than one sample containing protein, (a) labeling a peptide present in the first sample with a first isotope; b) labeling the peptides present in the second sample with a second isotope; (c) mixing the protein peptide mixture of the second sample with the protein peptide mixture of the first sample; ) Separating the combined protein-peptide mixture by chromatography into peptide fractions; (e) at least one amino acid of at least one of the peptides in each fraction chemically or enzymatically. , or chemically and enzymatically altered, thereby forming a peptide flagged And (f) isolating flagged peptides from each fraction by chromatography, wherein the chromatography is performed using the same type of chromatography as in step (d); g) performing mass spectrometric analysis of the isolated flagged peptides; (h) comparing the peak heights of the flagged peptides that are identical but differentially labeled with an isotope. Calculating the relative amount of flagged peptides in each sample, and (i) determining the identity of the flagged peptides and their corresponding proteins. 19. A method according to claim 18, wherein there is one or more pretreatment steps prior to step (d). 20. A method according to claim 18 or 19, wherein the chromatographic conditions of steps (d) and (f) are the same or substantially similar. Determination of identity of flagged peptides is performed by a method selected from the group consisting of tandem mass spectrometry combined with database search, post-source degradation analysis, peptide mass measurement and amino-terminal peptide mass measurement 21. The method of claim 18, 19 or 20, wherein: Determining the identity of the flagged peptide further comprises: (a) the presence of a modified amino acid; (b) the determination of the number of free amino groups in the flagged peptide; (c) a protein peptide mixture 23. The method of claim 21, wherein the method is based on knowledge of the splitting specificity of the protease used to generate the lysate; and (d) one or more of the large averages of the hydrophobicities of the peptides. In a method for determining the amount of at least one protein present in a sample, (a) preparing a protein peptide mixture; (b) differentially isotopically labeled compared to its reference peptide counterpart. Adding at least one synthetic reference peptide in a known amount to the mixture, whereby the synthetic reference peptide and the reference peptide counterpart are modified in step (d); (c) the mixture by chromatography (D) at least one amino acid of at least one of the peptides in each fraction is chemically, enzymatically, or chemically and enzymatically modified ; step of forming a peptide flagged by; (e) chromatogram Isolating flagged peptides from each fraction by the step, wherein the chromatography is performed in the same type of chromatography as in step (c); (f) Performing an analysis by mass spectrometry; (g) calculating the amount of protein present in the sample by comparing the peak height of the synthetic reference peptide to the reference peptide; and (h) the reference peptide and Determining the identity of the corresponding protein. 24. The method of claim 23, wherein there is one or more pretreatment steps prior to step (c). 25. A method according to claim 23 or 24, wherein the chromatographic conditions of steps (c) and (e) are the same or substantially similar. Determination of the identity of the reference peptide is performed by a method selected from the group consisting of tandem mass spectrometry combined with database searching, post-source degradation analysis, peptide mass measurement and amino-terminal peptide mass measurement. 26. A method according to claim 23, 24 or 25. The step of determining the identity of the reference peptide is further used to generate (a) the presence of a modified amino acid; (b) the number of free amino groups in the reference peptide; (c) a protein peptide mixture. 27. The method of claim 26, wherein said method is based on knowledge of the splitting specificity of the protease to be obtained; and (d) one or more of the large averages of the hydrophobicity of the peptide. In a method for isolating a subset of peptides from a protein peptide mixture,
(A) separating the protein-peptide mixture into peptide fractions by chromatography; (b) at least one amino acid of most of the peptides in each fraction chemically, enzymatically, or chemically manner and are enzymatically modified, step for generating a subset of unaltered identification peptides, and (c) or from each fraction of the unmodified by chromatography comprising the step of isolating the constant peptide, step (a ) And (c) chromatography is carried out with the same type of chromatography. 29. The method of claim 28, wherein the chromatographic conditions of steps (a) and (c) are the same or substantially similar. 30. A method according to claim 28 or 29, wherein there is one or more pretreatment steps prior to step (a). The method according to claim 28, 29 or 30, wherein the identification peptide is a peptide whose amino terminus is blocked. 31. The method of claim 28, 29 or 30, wherein the identification peptide is an amino terminal peptide. 31. The method of claim 28, 29 or 30, wherein the identification peptide is a carboxy terminal peptide. 31. A method according to claim 28, 29 or 30, wherein the identifying peptide is a peptide that has been internally proteolytically processed. 35. The method of claim 32, wherein the method distinguishes between an amino terminal peptide blocked in vivo and a free amino terminal peptide that is not blocked in vivo. 36. The method of any one of claims 28-35, further comprising identifying the identified peptide and its corresponding protein. 37. The method of claim 36, wherein the identifying step is assisted by a tandem mass spectrometer in combination with a database search. 37. The method of claim 36, wherein the identifying step is assisted by post source decomposition analysis in combination with a database search. 37. The method of claim 36, wherein the identifying step is performed by measuring peptide mass in combination with a database search. The identification step further comprises (a) determining the number of free amino groups in the identified peptide; (b) knowledge of the split specificity of the protease used to produce the protein peptide mixture; 40. The method of claim 39, wherein the method is based on one or more of the large averages of fobicity. (B) labeling a peptide present in a first sample with a first isotope in a method for determining the relative amount of at least one protein in a plurality of samples containing proteins; ) Labeling the peptides present in the second sample with a second isotope; (c) mixing the protein peptide mixture of the second sample with the protein peptide mixture of the first sample; (d) Separating the combined protein-peptide mixture into peptide fractions by chromatography; (e) chemically or enzymatically, or at least one amino acid of most of the peptides in each fraction; step of forming identification peptide thereby chemically and is enzymatically modified, and (f) chromatogram Isolating the identified peptide from each fraction by a fee, wherein the chromatography is performed in the same type of chromatography as in step (d); (g) mass spectrometry of the isolated identified peptide Performing an analysis according to: (h) calculating the relative amount of identified peptides in each sample by comparing the peak heights of identical but differentially isotopically labeled identified peptides; and (I) A method comprising determining the identity of the identified peptide and its corresponding protein. 42. The method of claim 41, wherein there is one or more pretreatment steps prior to step (d). 43. The method of claim 41 or 42, wherein the chromatographic conditions of steps (d) and (f) are the same or substantially similar. Determination of the identity of the identified peptide is performed by a method selected from the group consisting of tandem mass spectrometry combined with database search, post-source degradation analysis, peptide mass measurement and amino-terminal peptide mass measurement. 44. A method according to claim 41, 42 or 43. Determining the identity of the identity peptide further comprises (a) determining the number of free amino groups in the identity peptide; (b) knowledge of the split specificity of the protease used to produce the protein peptide mixture; 45. The method of claim 44, wherein (c) is based on one or more of the large averages of peptide hydrophobicities. In a method for determining the amount of at least one protein present in a sample, (a) preparing a protein peptide mixture; (b) differentially isotopically labeled compared to its reference peptide counterpart. Adding at least one synthetic reference peptide in a known amount to the mixture, whereby the synthetic reference peptide and the reference peptide counterpart are modified in step (d); (c) the mixture by chromatography (D) at least one amino acid of most of the peptides in each fraction is chemically, enzymatically, or chemically and enzymatically modified thereby step of forming identification peptide; (e) each hula chromatography Isolating the identified peptide from the step, wherein the chromatography is performed with the same type of chromatography as in step (c); (f) performing mass spectrometry analysis of the identified peptide; (G) calculating the amount of protein present in the sample by comparing the peak height of the synthetic reference peptide with its reference peptide counterpart; and (h) determining the identity of the reference peptide and its corresponding protein. A method comprising the step of: 47. The method of claim 46, wherein there is one or more pretreatment steps prior to step (c). 48. The method of claim 46 or 47, wherein the chromatographic conditions of steps (c) and (e) are the same or substantially similar. Determination of the identity of the reference peptide is performed by a method selected from the group consisting of tandem mass spectrometry combined with database searching, post-source degradation analysis, peptide mass measurement and amino-terminal peptide mass measurement. 49. A method according to claim 46, 47 or 48. Determining the identity of the reference peptide further comprises (a) determining the number of free amino groups in the reference peptide; (b) knowledge of the split specificity of the protease used to generate the protein peptide mixture; 50. The method of claim 49, wherein (c) is based on one or more of the large average hydrophobicity of the reference peptide. Primary chromatography column for separating a protein-peptide mixture into multiple fractions under a defined set of conditions, whereby each fraction is subsequently subjected to at least one amino acid modification to produce a flagged peptide And the modified fractions are pooled into a set of pooled fractions, each of the pooled fractions including a primary chromatography column containing at least two modified fractions; and a first pooled fraction A first secondary chromatography column for separation and at least a second secondary chromatography column arranged in parallel with the first secondary chromatography column for separating a second pooled fraction Secondary chromatograph -Isolation of flagged peptides under a set of columns that are substantially identical to a defined set of conditions, thereby (i) flags from different fractions within or between pools And (ii) a secondary chromatography column set with no elution overlap between the flagged and unmodified peptides, and a system for sorting the peptides. A primary chromatography column for separating a protein-peptide mixture into a plurality of fractions under a defined set of conditions, whereby each fraction is subsequently modified with at least one amino acid modification and an unmodified peptide A primary chromatography column that produces the identified peptide and the modified fractions are pooled into a set of pooled fractions, each pooled fraction containing at least two modified fractions; and a first pool A first secondary chromatography column for separating the separated fractions and at least a second secondary chromatography arranged in parallel with the first secondary chromatography column for separating the second pooled fractions Including a chromatography column Chromatographic column set isolation of identified peptides under conditions that are substantially identical to a defined set of conditions, thereby (i) identifying from different fractions within or between pools A system for sorting peptides comprising: (ii) a secondary chromatography column set with no elution overlap between the identified peptide and the modified peptide. 53. The method according to any one of claims 51 to 52 , further comprising an outlet to a second chromatography column set for recovering eluates from the first secondary chromatography column and the second secondary chromatography column. The described system. 54. The system of claim 53 , further comprising an analyzer coupled to the outlet. 54. The system of claim 53 , further comprising a waste container coupled to an outlet for recovering waste from the secondary chromatography column set. 53. The sample injector further comprising a sample injector coupled to a secondary chromatography column set for injecting a pooled fraction into one of the first secondary column and the second secondary column. The described system. 57. The system of claim 56 , further comprising a sample injection valve set for directing a pooled fraction from the sample injector to one of the first secondary column and the second secondary column. 53. The system of claims 51-52 , further comprising a solvent system for providing a solvent gradient for the secondary chromatography column set. A solvent system provides a first solvent pump for providing a solvent gradient for the first secondary chromatography column and a second solvent for providing a solvent gradient for the second secondary chromatography column 59. The system of claim 58 , comprising a pump. A solvent pump coupled to the first secondary chromatography column and the second secondary chromatography column; a first flow rate adjustment for adjusting a solvent flow rate to the first secondary chromatography column; 59. The system of claim 58 , comprising a second flow rate regulator for regulating the solvent flow rate to the vessel and the second secondary chromatography column. 53. The system of claims 51-52 , further comprising a fraction collector for recovering eluate from the secondary chromatography column set. 58. The system of claim 57 , further comprising a valve control system for controlling the sample injection valve set. 53. The system of claims 51-52 , wherein the first and second secondary chromatography columns are substantially identical to the primary column. A first solvent gradient was applied to the primary column, resulting in the separation of the protein peptide mixture, and a second solvent gradient that was substantially identical to the first solvent gradient was applied to the secondary column and pooled 53. A system according to claims 51 to 52 , wherein separation is provided for fractionation. (A) providing a primary chromatography column for separating the protein peptide mixture; (b) the protein peptide in the primary chromatography column to separate the protein peptide mixture into a set of fractions under a defined set of conditions. Injecting the mixture; (c) modifying at least one of the fractions in the fraction set to form a set of modified fractions, wherein the modified fractions of the flagged peptides Comprising a subset and a subset of unmodified peptides; (d) pooling the first modified fraction and the second modified fraction to form a first pooled fraction, wherein i) FF from the first and second modified fractions Ii) no elution overlap between the unmodified peptide of the fraction and the flagged peptide; (e) the third modified fraction and the fourth modification. Pooling the fractions formed to form a second pooled fraction, wherein i) between the flagged peptides from the third and fourth modified fractions, and ii) said (F) a first secondary chromatography to separate a flagged subset of peptides from a subset of unmodified peptides, wherein there is no elution overlap between the fractions of unmodified peptides and flagged peptides; Providing a column; and (g) first pooled using a secondary chromatography column under a defined set of conditions. Separating the fraction and isolating the flagged peptide from the protein peptide mixture comprising isolating the flagged peptide subset in the first modified fraction and the second modified fraction Method. (A) providing a primary chromatography column for separating the protein peptide mixture; (b) injecting the protein peptide mixture into the primary chromatography column and subjecting the protein peptide mixture to a set of fractions under a defined set of conditions. (C) modifying at least one of the fractions in the fraction set to form a set of modified fractions, wherein the modified fraction is a subset of the modified peptides And (d) pooling the first modified fraction and the second modified fraction to form a first pooled fraction, wherein i) A modified page from the first and second modified fractions. And ii) no elution overlap between the identified peptide of the fraction and the modified peptide; (e) pooling the third modified fraction and the fourth modified fraction; Forming a second pooled fraction, wherein i) between the identified peptides from the third and fourth modified fractions and ii) the identified peptides and modified peptides of said fractions (F) providing a first secondary chromatography column to separate the modified subset of peptides from the identified subset of peptides; and (g) the specified conditions The first pooled fraction is separated using a secondary chromatography column under the set and the first modified fraction is separated. Comprising the step of isolating a subset of identification peptides with transfection second modified in fractions, the method of isolating the identification peptides of protein peptide mixture. Peptides that separated the second pooled fraction using the first secondary chromatography column under a defined set of conditions and flagged the third modified fraction and the fourth modified fraction 67. The method of claim 65 or 66 , further comprising isolating a subset of the identified peptides. (H) a second secondary chromatography column arranged substantially in parallel with the first secondary chromatography column for separating the modified peptide subset from the subset of unmodified peptides in the fraction; And (i) separating the second pooled fraction using a second secondary chromatography column under a defined set of conditions, the third modified fraction and the second modified fraction 67. The method of claim 65 or 66 , further comprising isolating a subset of the modified peptides in the fraction. 67. The method of claim 65 or 66 , further comprising the step of directing the flagged or identified peptide to an analyzer. 70. The method of claim 69 , further comprising identifying the identified peptide or flagged peptide and its corresponding protein using an analytical device in combination with a database search.

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